AN ABSTRACT OF THE DISSERTATION OF
Aishwarya V. Ramaswamy for the degree of Doctor of Philosophy in Microbiology
presented on June 02, 2005.
Title: Cloning and Biochemical Characterization of the Hectochiorin Biosynthetic
Gene Cluster from the Marine Cyanobacterium Lyngbya maluscula
Abstract approved:
Redacted for privacy
William H. Gerwick
Cyanobacteria are rich in biologically active secondary metabolites, many of
which have potential application as anticancer or antimicrobial drugs or as useful
probes in cell biology studies. A Jamaican isolate of the marine cyanobacterium,
Lyngbya majuscula was the source of a novel antifungal and cytotoxic secondary
metabolite, hectochlorin. The structure of hectochiorin suggested that it was derived
from a hybid PKS/NIRPS system. Unique features of hectochlorin such as the
presence of a gem dichloro functionality and two 2,3-dihydroxy isovaleric acid
prompted efforts to clone and characterize the gene cluster involved in hectochiorin
biosynthesis.
Initial attempts to isolate the hectochlorin biosynthetic gene cluster led to the
identification of a mixed PKS/NRPS gene cluster, LMcryl , whose genetic architecture
did not substantiate its involvement in the biosynthesis of hectochlorin. This gene
cluster was designated as a cryptic gene cluster because a corresponding metabolite
remains as yet unidentified. The expression of this gene cluster was successfully
demonstrated using RT-PCR and these results form the basis for characterizing the
metabolite using a novel interdisciplinary approach.
A 38 kb region putatively involved in the biosynthesis of hectochiorin has also
been isolated and characterized. The hct gene cluster consists of eight open reading
frames (ORFs) and appears to be colinear with regard to hectoclilorin biosynthesis.
An unusual feature of this gene cluster includes the presence of a ketoreductase
domain in an NRPS module and appears to be the first report of such an occurrence in
a cyanobacterial secondary metabolite gene cluster Other tailoring enzymes present
in the gene cluster are two cytochrome P450 monooxygenases and a putative
halogenase. The juxtaposition of two ORF's with identical modular organization
suggests that this gene cluster may have resulted from a gene duplication event.
Furthermore, biochemical characterization of two adenylation domains from this
cluster strengthens its involvement in the biosynthesis of hectochiorin.
©Copyright by Aishwarya V. Ramaswamy
June 02, 2005
All Rights Reserved
Cloning and Biochemical Characterization of the Hectochiorin Biosynthetic Gene
Cluster from the Marine Cyanobacterium Lyngbya
by
Aishwarya V. Ramaswamy
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented June 02, 2005
Commencement June 2006
majuscula
Doctor of Philosophy dissertation of Aishwarya V. Ramaswamy presented on June 02,
2005.
APPROVED:
Redacted for privacy
Maj or Professor, representing Microbiology
Redacted for privacy
Chair of the Department of Microbiology
Redacted for privacy
Dean of the GiIaduè School
I understand that my dissertation will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes the release of my
dissertation to any reader upon request.
Redacted for privacy
shwarya V. Ramaswamy, Author
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my major professor, Dr.
William H. Gerwick, for his remarkable support and guidance throughout the course
of my graduate work. I would also like to thank my committee members, Dr. Mark
Zabriskie, Dr. Steve Giovannoni, Dr. George Constantine and Dr. Jeff Stone for their
assistance.
11 am very grateful to Dr. Patricia Flatt and Dr. Daniel Edwards for their
mentorship and invaluable guidance throughout my graduate career. I would like to
acknowledge Dr. Kerry McPhail, Dr. Harald Gross and Dr. Lena Gerwick for their
academic guidance. I would also like to thank Gloria Zeller for her help and
friendship. Special thanks also go to former and current members of the Gerwick
group for their friendship.
Most importantly, I share this accomplishment with my parents and am deeply
indebted to them for their love, constant encouragement, unconditional support and
confidence in my abilities. Finally, I thank my husband for his love and patience
which helped me immensely during these challenging years.
TABLE OF CONTENTS
CHAPTER I: GENERAL INTRODUCTION .........................................
1
GENERAL THESIS CONTENTS ...........................................
38
REFERENCES ..................................................................
39
CHAPTER II: ISOLATION OF A CRYPTIC GENE CLUSTER
FROM THE CYANOBACTERIUM LYNGBYA
MAJUSCULA JHB AND DEMONSTRATION OF
ITS EXPRESSION
INTRODUCTION .............................................................
54
RESULTS AND DISCUSSION..............................................
57
EXPERIMENTAL ..............................................................
85
REFERENCES .................................................................
92
CHAPTER III: CLONING AND BIOCHEMICAL
CHARACTERIZATION OF THE HECTOCHLOR1N
BIOSYNTHETIC GENE CLUSTER FROM THE
MARINE CYANOBACTERIUM LYNGBYA
MAJUSCULA
INTRODUCTION ..............................................................
99
RESULTS AND DISCUSSION .............................................
102
.............................................................
126
REFERENCES ......................................................................
132
EXPERIMENTAL
TABLE OF CONTENTS (Continued)
CHAPTER IV: EXPRESSION AND BIOCHEMICAL
CHARACTERIZATION OF ADENYLATION
DOMAINS FROM THE CARMABIN
BIOSYNTHETIC GENE CLUSTER
INTRODUCTION................................................................................
138
RESULTS AND DISCUSSION...........................................................
143
EXPERIMENTAL ...............................................................................
148
REFERENCES.....................................................................................
150
CHAPTER V: CONCLUSIONS ..........................................................................
153
BIBLIOGRAPHY ................................................................................................
156
LIST OF FIGURES
Figure
Page
1.1
NRPS module organization...............................................................
20
1.2
Recognition of cognate amino acid by binding pocket
residues (A) and activation of amino acid by the NIRPS A-domain
(B) ......................................................................................................
21
1.3
Post-translational modification of the PCP domain ..........................
22
1.4
Loading of PCP domain with activated amino acid..........................
22
1.5
Condensation domains catalyze peptide bond formation .................. 23
1.6
Steps involved in nonribosomal peptide biosynthesis ....................... 24
1.7
Reactions catalyzed by NRPS tailoring domains ..............................
1.8
PKS module organization .................................................................. 28
1.9
Recognition of substrate by the AT domain and its
transfer to ACP domain ..................................................................... 29
1.10
Ketide bond formation catalyzed by the KS domain........................
29
1.11
Reactions catalyzed by PKS auxiliary domains ................................
30
1.12
The barbamide (53) biosynthetic gene cluster ..................................
35
25
LIST OF FIGURES (Continued)
Figure
1.13
The curacin A (14) biosynthetic gene cluster....................................
35
1.14
The jamaicamide A (27) biosynthetic gene cluster...........................
36
1.15
The lyngbyatoxin A (30) biosynthetic gene cluster..........................
36
ILl
Isolation of high molecular weight genomic DNA...........................
58
11.2
Proposed biosynthetic precursors of hectochiorin ............................
60
11.3
Amplification of NRPS A-domain probes ........................................
62
11.4
ClustalW alignment of cysteine adenylation domains ......................
63
11.5
Amplification of cyteine-specific A-domain ..................................... 64
11.6
Amplification of KS domain probe ...................................................
65
11.7
Restriction digest analyses (A) and Southern hybridizations of
cosmids 1-105 and 111-105 (B) ...........................................................
67
Genetic architecture of LMcryland modular organization of
LMcryl ..............................................................................................
70
Alignment of heterocyclization domains ..........................................
71
11.8
11.9
LIST OF FIGURES (Continued)
Figure
11.10
Alignment of N-methyl transferases .................................................
73
11.11
Nonribosomal peptides from freshwater and terrestrial
cyanobacteria .....................................................................................
73
Sequence alignments of PKS domains of LMcryl with other
cyanobacterial PKS's ........................................................................
75
Structure of proposed peptide encoded by hybrid NRPS/PKS
encoded by LMciyl. R = unknown side chain..................................
76
Reversed phase HPLC profiles for the crude extract and nine
normal phase VLC fractions (A-I) of the cultured Lyngbya
majusculaJHB ..................................................................................
79
Schematic representation of the novel approach used to
isolate and elucidate structure of putative metabolite encoded by
LMcryl ..............................................................................................
80
11.16
Primers for RT-PCR .........................................................................
82
11.17
Results of RT-PCR ...........................................................................
83
111.1
Chemical structure of hectochiorin (54) and related metabolites
101
111.2
High molecular weight genomic DNA from cultured L. majuscula
JHB ...................................................................................................
102
Alignment of heterocyclization-adenylation domains for primer
design (A) and PCR amplification of 1.8 kb heterocyclizationadenylation domain probe (B) ..........................................................
104
11.12
11.13
11.14
11.15
111.3
LIST OF FIGURES (Continued)
Figure
ge
111.4
Genetic map of the hct gene cluster spanning
38 kb .....................
106
111.5
Alignment of barbamide BarB 1 and B2, syringomycin
SyrB2, coronamic acid CmaB and PAHX with HctB ......................
108
Sequence alignments of PKS domains from HctD with other
cyanobacterial PKS's ........................................................................
109
Overexpression and purification of adenylation domains in HctE
and Western blot analysis .................................................................
112
Results of the ATP-PPi exhange assay for HctEAO.IVA (A) and
HctEAc (B) and represent average of duplicate experiments .........
114
111.9
Overexpression and purification of HctA .........................................
115
111.10
The proposed biosynthesis of hectochiorin......................................
116
111.11
Alignment of heterocyclization domains ..........................................
123
IV.1
Genetic architecture of the carmabin A biosynthetic gene
cluster ................................................................................................
140
IV.2
Overexpression of CarG (A) and Carl (B) adenylation domains
145
IV.3
Results for the ATP-PPi exhange assay for CarG A-domain (A)
and Carl A-domain (B) .....................................................................
147
111.6
111.7
111.8
LIST OF TABLES
Table
Page
List of selected marine-derived natural products in clinical and
preclinicalstudies ..............................................................................
3
11.1
Substrate specificity of NRPS A-domain probes ..............................
62
11.2
Substrate specificity of cysteine A-domain probe .............................
64
11.3
Substrate specificities of A domain LMcryl and other related Adomains ..............................................................................................
74
Deduced functions of the proteins in the hct biosynthetic gene
cluster.................................................................................................
107
Substrate specificity of A-domains from the first modules in HctE
andHctF ............................................................................................
119
Substrate specificity of the adenylation domains in the second
modules present in HctE and HctF ....................................................
122
IV.1
Deduced functions of open reading frames in the car gene cluster...
140
IV.2
Substrate specificity of A-domains in car .........................................
142
1.1
111.1
111.2
111.3
LIST OF ABBREVIATIONS
A
Adenylation
aa
Amino acid
ACP
Acyl carrier protein
AMP
Adenosine monophosphate
AT
Acyl transferase
ATP
Adeno sine triphosphate
BLAST
Basic alignment search tool
bp
Base pairs
C
Condensation
cDNA
Complementary DNA
CTAB
Hexadecyltrimethylammonium bromide
DII
Dehydratase
DHiv
2,3-dihydroxyisovaleric acid
DIG
Digoxigenin
DNA
Deoxyribonucleic acid
DTT
Dithiothreitol
E
Epimerase
EDTA
Ethylenediaminetetraacetic acid
ER
Enoyl reductase
GHNMQC
Gradient hydrogennitrogen multiple quantum coherence
Hal
Halogenase
Het
Heterocyclization domain
His
Histidine
kb
kilobase
kDa
kilodalton
KR
Ketoreductase
KS
Ketosynthase
LIST OF ABBREVIATIONS (Continued)
mRNA
messenger RNA
MT
Methyl transferase
NCBI
National Center for Biotechnology Information
NMR
Nuclear magnetic resonance
N-Mt
N-methyl transferase
NRPS
Nonribosomal peptide synthetase
0-IVA
2-oxo-valine
2-OH WA
2-hydroxy isovaleric acid
ORE
Open reading frame
Ox
Oxidase
PAGE
Polyacrylamide gel electrophoresis
PCP
Peptidyl carrier protein
PCR
Polymerase chain reaction
PKS
Polyketide synthase
Ppant
Phosphopantetheine
Ppi
Inorganic pyrophosphate
RNA
Ribonucleic acid
RT
Reverse transcriptase
RT-PCR
Reverse transcriptase-polymerase chain reaction
SAM
S-adenosyl methionine
SDS
Sodium dodecyl sulphate
TE
Thioesterase
VLC
Vacuum liquid chromatography
CLONING AND BIOCHEMICAL CHARACTERIZATION OF THE
HECTOCHLORIN BIOSYNTHETIC GENE CLUSTER FROM THE MARINE
CYANOBACTERIUM LYNGBYA MAJUSCULA
CHAPTER I
GENERAL INTRODUCTION
Marine natural products
Nature has been a source of medicinal agents for thousands of years and the
use of natural products in the treatment of human diseases has been well documented
in both traditional Eastern and Western medicine (Newman et al., 2000). Natural
products have played a significant role in the process of drug discovery and
development and about 60% of drugs in current use are derived from natural products
(Newman et al., 2003). The inexorable increase in the emergence of drug-resistant
microbes and parasites in recent decades has created a pressing need for the
development of new therapeutics (Dougherty et al., 2002). Additionally, the discovery
of new pharmaceuticals is vital for treatment of other diseases such as inflammation,
immune disorders and different types of cancer. Terrestrial plants and
microorganisms have been long recognized as a valuable source of bioactive
compounds and serve as an important source for the discovery of new
pharmaceuticals. In fact, soil actinomycetes account for the production of over 7000
bioactive compounds (Jensen et al., 2005). Lately, the marine environment, which has
remained relatively unexplored, is beginning to gain much attention as a reservoir of
bio active natural products.
In contrast to studies on terrestrial natural products, exploration of marine
natural products started 50 years ago (Bergman and Feeney, 1951) and it was only in
the 1970's that an intensive search for natural products from marine plants and
invertebrates was initiated. Several factors spurred this vigorous search for bioactive
2
natural products from the sea. The oceans comprise over 70% of the earth's surface
and there exists a wealth of biodiversity in the marine environment. Conservative
estimates suggest that there are over ten million species and a majority of these remain
undescribed (Grassle and Maciolek, 1992; May, 2005). Recently, whole-genome
shotgun sequencing of microbial populations from the Sargasso Sea near Bermuda
revealed the presence of enormous diversity and abundance of organisms and is
believed to represent only a fraction of entire population (Venter et al., 2004). The
presence of such a tremendous variety of flora and fauna in the marine ecosystem
along with distinct physicochemical conditions present in the ocean create an
intensively competitive environment which in turn fosters the development of a wide
range of survival strategies not found among terrestrial organisms. As such, natural
products produced by marine organisms exhibit tremendous structural complexity and
possess novel functionalities and appear to be highly evolved as potent inhibitors of
physiological processes in potential predators. This is especially true for sessile
invertebrates such as sponges and tunicates that produce toxic metabolites to ward off
potential predators. Thus, the marine realm represents a wealth of resource for natural
product chemistry and biotechnology.
At present, approximately 16,000 natural products have been characterized
from marine organisms (MarineLit, 2005). Marine microorganisms, phytoplankton,
algae, sponges, soft corals and tunicates are among the predominant sources of novel
chemical entities (Blunt et al., 2005). A considerable number of natural products
isolated from marine organisms appear to possess anticancer activity and several
candidates are currently in clinical studies or preclinical trials. Additionally, some
marine-derived bioactive compounds are also being developed as pain-killers and antiinflammatory substances (Donia and Hamann, 2003). Table 1.1 adapted from several
recent reviews on marine-derived lead compounds (Newman and Cragg, 2004b;
Newman and Cragg, 2004a; Jimeno et al., 2004; Simmons et al., 2005) provides
examples of notable natural products of marine origin or their synthetic derivatives
that are currently in use or in preclinical/clinical studies.
3
Table 1.1: List of selected marine-derived natural products in clinical and
preclinical studies.
Name
Ara-C (1)
Ara-A (2)
Ecteinascidin (3)
(ET743)
(Rinehart et al., 1990).
Ziconotide (4)
(Ferber et al., 2003)
Bryostatin (5) (Pettit et
al., 1982).
Aplidine (6)
(Rinehart and
Lithgow-Bertelloni,
Source
Anticancer (acute myelocytic
turbinata
It has been shown to bind to the minor
groove of DNA but downstream
mechanisms still unclear but may
interfere with DNA binding proteins
and transcription. Also shown to affect
nucleotide excision repair (Manzanares
et al., 2001).
Conus magus
Neuropathic pain
Clinical use
Bugula neritina
Anticancer
Phase II
Antiviral (Infectious herpes)
Clinical use
Anticancer
Phase III
Aplidium
Anticancer
albicans
Blocks the secretion of angiogenic
factor vascular endothetial growth
factor and may have an effect on tumor
angiogenesis. The exact mechanism
remains to be clarified (Taraboletti et
al., 2004).
Elysia rufescens/
Bryopsis sp.
Anticancer
Cyanobacterial
symbiont of
Dolabella
Anticancer
auricularia
TZT- 1027 (9)
(Miyazaki et al.,
1995).
Clinical use
leukemia)
Serves as a competitive inhibitor of
tumor-promoting phorbol esters by
binding to identical receptor sites on
protein kinase C without possessing
tumor promoting activity itself
(Amador et al., 2003).
1993).
Dolastatin 10 (8)
(Pettit et al., 1987).
Status
Synthetic analog
of sponge
nucleosides
Synthetic analog
of sponge
nucleosides
Ecteinascidia
1991).
Kahalalide F (7)
(Hamann and Scheuer,
Target/iViechanism of action
Synthetic
dolastatin
Phase II
Phase II
Selective for tumors with high
lysosomal activity. Found to induce
cell-death probably initiated by
lysosomal membrane polarization
(Garcia-Rocha et al., 1996).
Binds to tubulin and inhibits
microtubule assembly and subsequently
blocks cytokinesis. Also shown to be a
non-competitive inhibitor of Vinca
alkaloids (Bai et al., 1990).
Anticancer
The synthetic derivative of dolastatin
10 was designed to have enhanced
antitumor activity and reduced
cytotoxic effects (Kobayashi et al.,
1997).
Phase II
Table I.! continued.
Name
Discodermolide (10)
(Gunasekera et al.,
Source
Target/Mechanism of action
Discodermia
dissoluta
Anticancer
E73 89 (11)
Lissodendoryx
Anticancer
(Halichondrin B
derivative) (Aicher et
al., 1992).
IPL-576092 (12)
Derivative of
contignasterol
(Burgoyne et al.,
sp.
Binds tubulin at a site distinct from the
Vinca site and causes tubulin
depolymerization (Towle et aL, 2001).
Petrosia contignata
Anti-inflammatory
1990).
Phase I
Microtubule stabilizing agent which
causes arrest cell cycle arrest at the
G2IM phase. Also possesses
immunosuppressive properties
(Kalesse, 2000).
Phase I
Phase I
It inhibits allergen-induced histamine
release from mast cells. Topical
application showed that it inhibited
allergen-induced plasma protein
exudation (Coulson and ODonnell,
2000).
1992).
Eleutherobin (13)
(Lindel et al., 1997).
Eleutherobia sp.
Curacin A (14)
(Gerwick et al., 1994).
Lyngbya ma] uscula
Thiocoraline (15)
(Perez-Baz et al.,
1997; Romero et al.,
Micromonospora
marina
Anticancer
Salinospora sp.
Proteosome inhibitor
Anticancer
Preclinical
Induces tubulin polymerization in vitro
in a manner similar to paclitaxel. Cells
exposed to eleutherobin contain
multiple nuclei and arrest at mitosis
(Long et al., 1998).
Anticancer
Preclinical
Binds to colchicine site of tubulin and
induces depolymerization induced by
glutamate or microtubule associated
proteins (Wipfet al., 2004).
1997).
Salinosporamide A
(16) (Fehling et al.,
2003).
Status
Preclinical
Thiocoralme inhibits DNA elongation
and probably inhibits the activity of
DNA polymerase-a (Erba et at., 1999).
(salinosporamide A inhibited
proteasomal chymotrypsinlike proteolytic activity)
Also displayed in vitro cytotoxicity
against several cancer cell lines
(Fehling et al., 2003).
Preclinical
NH2
NH2
NN
N
ON
HO0
HOOI
HO
HO
OH
OH
Ara-A (2)
Ara-C (1)
cidin 743 (3)
NH2-CKGKGAKCSRLMYDC TGSCRSGKC-CONH2
Ziconotide (4)
OH 0
NH
00
N
Bryostatin 1 (5)
H2N
NNH
OCH3
Aplidine (6)
0NH
O
HN
0
O2NH
H
0
/
HN
'H
0
0
HO
Y H
T0
NH
\
/
O
0
N
TOCH ( TOCHy
Dolastatin 10 (8)
Kahalalide F (7)
0
NANTIrIyNO
..-
CH3OH 0
0CH
TZT 1027 (9)
OH
OH
NH2
Discodermoljde (10)
H2N
HOJOH
E7389 (11)
0
IPL 576092 (12)
o:
CH3
OCH3
O OCH3
N4,,CH3
OCH3
CuracinA (14)
OH
Eleutherobin (13)
QoH
O
N
NO
ONH
HN
O
H
sS
o
0
Thiocoraline (15)
O
ci
NH0
HO-
Salinosporamide A (16)
H'
V
'H
7
The preceding section has illustrated some of the significant contributions of
marine natural products especially to the field of biomedicine. Among marine
organisms, cyanobacteria are emerging as one of the most prolific producers of novel
bioactive secondary metabolites (Burja et al., 2001; Gerwick et al., 2001). In the
following section, a brief overview of cyanobacteria will be presented along with
examples of some noteworthy secondary metabolites isolated from marine
cyanobacteria (especially those belonging to the genus
Lyngbya).
A predominant
theme encountered in cyanobacterial secondary metabolites is the rich integration of
complex enzyme systems known as polyketide synthases and nonribosomal peptide
synthetases. Details regarding these enzymes and the biosynthetic gene clusters of
notable cyanobacterial metabolites will be discussed in the next section. The final
section will provide a brief outline of the various chapters presented in this thesis.
Secondary metabolites from marine cyanobacteria
Cyanobacteria, also known as blue-green algae, are ancient (3.5 Ma years) and
morphologically the most diverse photoautotrophic prokaryotes, which inhabit a wide
diversity of habitats including open oceans, tropical reefs, shallow water
environments, terrestrial substrates such as rocks, aerial environments such as in trees,
and fresh water ponds, streams and puddles. Most cyanobacteria are generalists and
can tolerate a great range of environmental conditions (Whitton and Potts, 2000). It is
presumed that cyanobacteria evolved in the Precambrain Era and caused the transition
in the Earth's atmosphere from its primeval anaerobic state to its current oxygenic one
(Schopf, 2005). The first global evolutionary scheme of cyanobacteria based on
phylogenetic analysis of 1 6s rRNA indicated that they underwent rapid diversification
within a relatively short span of time (Giovannoni et al., 1988). Cyanobacteria were
regarded as algae by botanists until electron microscopy and other biochemical studies
provided conclusive evidence in favor of their prokaryotic nature. Therefore, early
cyanobacterial taxonomy was based on morphological features in accordance to the
Botanical code. However, this approach was confronted by Stanier and collaborators
who pioneered the use of physiological and genotypic features determined with axenic
cultures and advocated a bacteriological approach for cyanobacterial taxonomy
(Stanier et al., 1978). The basis of bacteriological taxonomy of cyanobacteria was
first proposed by Rippka (Rippka et al., 1971). This system also relies on morphology
to a large extent and allows identification of strains at the generic level. A modified
version of this system is described in Bergey's Manual of Systematic Bacteriology
[Subsection I (Chroococcales), Subsection II (Pleurocapsales), Subsection III
(Oscillatoriales), Subsection IV (Nostocales) and Subsection V (Stigonematales)]
(Castenholz et al., 2001). While analyses with 16S rDNA sequences have revealed
that two out of the five taxanomical groupings are phylogenetically coherent, members
belonging to Chroococcales, Pleurocapsales and Oscillatoriales do not form coherent
phylogenetic lineages making phylogenetic relationship among cyanobacteria
disputatious (Giovannoni et al., 1988; Wilmotte, 1994; Ishida et al., 2001; Seo and
Yokota, 2003). Although 16S rDNA sequence analyses have provided valuable
information on the genotypic relationships of cyanobacteria, a polyphasic approach to
link this molecular data with phenotypic studies is important in order to obtain a more
meaningful interpretation of these results.
Cyanobacteria play a major role in global cycles of several key elements and
have also been recognized as model systems for studying important physiological
processes such as photosynthesis (Barry et al., 1994) and circadian organization
(Iwasaki and Kondo, 2004). Cyanobacteria are becoming increasingly important
economically in both positive and harmful ways. They possess a rich assortment of
photosynthetic pigments such as chlorophyll a and b as well as phycoerythrin,
phycocyanin and allophycocyanin. Phycoerythrin present in cyanobacteria has found
application as a conjugate to antibodies that then allow visualization of cellular
constituents and processes (Batard et al., 2002) and chlorophyll is being explored for
its chemoprevention properties (Egner et al., 2001). Other cyanobacteria such as
Spirulina are being promoted as health food because they contain essential amino
acids. On the other hand, certain freshwater bloom-forming cyanobacteria
contaminate drinking water supplies by producing neurotoxins such as saxitoxin (17)
and hepatotoxins like microcystins (18) and nodularin (19) and pose significant threat
to human and livestock health (Briand et al., 2003). Cyanobacterial blooms have also
been shown to impact ecosystem structure and trophic interactions (Paerl, 1988).
H2NO
gJH
I
HN'N
)=NH
Nft0H
OH
Saxitoxin (17)
O
CH3O
COOH
NH
OCH3
H3C,
CH2I
o
HN
NN CH3
NH
HN
COO
NH
H3C0
H
cH3
0
H
H
CH3 CH3 _yNy1yN(L.L.CH
H2N-
,CH3
CH3 CH3 0NNH
H3C
CH3
COOH
COOH
NH
HN=<
NH2
Microcystin-LR (18)
Nodularin (19)
Marine cyanobacteria appear to possess an additional feature, the rich
elaboration of bioactive natural products that has enabled these organisms to survive
in predator-rich tropical reef ecosystems. Richard Moore at the University of Hawaii
pioneered the chemical investigation of marine cyanobacteria for their unique natural
products. In the early 1970's his laboratory published several surveys of marine
cyanobacteria from the Pacific showing that they were rich in potential anticancer and
antiviral substances (Moore, 1978; Moore, 1981; Moore, 1996). Early work showed
that cyanobacteria, as opposed to most other marine life forms at the time, were rich in
nitrogen-containing natural products. Subsequently, freshwater cyanobacteria were
shown to have similar biosynthetic capabilities and to produce an equally exciting
array of biologically active lipopeptides. They are now widely recognized to be one of
10
the richest of all marine life in this regard, both as free living organisms as well as
when living in association with marine invertebrates (sponges and tunicates) (Sings
and Rinehart, 1996; Gerwick et al., 2001; Piel, 2004).
As a result, members of the Oscillatoriaceae, (especially the filamentous
cyanobacterium Lyngbya majuscula) have proven to be exciting sources of novel
natural products with therapeutic and biotechnological potential. Over 200 novel
secondary metabolites have been isolated from various collections of L. majuscula
(MarinLit, 2005) and accounts for about 30% of all natural products reported from
marine cyanobacteria (Burja et al., 2001). Detailed experimental investigation of the
ecological role of marine cyanobacterial natural products has generally shown that
marine cyanobacteria are chemically defended against predation by generalist feeders
(Nagle and Paul, 1999; Nagle and Paul, 1998). Interestingly the chemical composition
of some marine cyanobacteria, especially Lyngbya majuscula, is remarkably diverse
between different populations. For instance, at least five different chemotypes were
identified from an apparent monoculture of L. majuscula from Guam (Nagle and Paul,
1999). A comparison of morphological characteristics, secondary metabolite
composition and 16S rDNA sequences revealed that morphology and secondary
metabolite profile could not be correlated to genetic variation and suggest that
phylogeny based on 1 6S rDNA cannot be used to predict chemical variability
(Thacker and Paul, 2004). The apparent lack of correlation could be a result of the
higher rate of evolution of secondary metabolite gene clusters or horizontal transfer of
these clusters between organisms. Alternately, the varied chemical profiles of
cyanobacteria may also be an adaptive response to the environmental conditions
present at a particular location (Nagle and Paul, 1999; Thacker and Paul, 2004).
A high proportion of cyanobacterial secondary metabolites appear to possess
the ability to interfere with the assembly of protein polymers in eukaryotic cells,
particularly, actin and tubulin. Another emerging trend is the isolation of metabolites
that target mammalian ion channels like the voltage-gated sodium channel. L.
majuscula is also notorious for the formation of massive blooms in recreational waters
often causing contact dermatitis and other skin irritations (Osborne et al., 2001). The
11
following section recounts several prominent marine cyanobacterial products based on
their biological activity.
i. Tubulin binding compounds
Microtubules play many significant roles in cell biology and microtubule
assembly is vital for the formation of spindle apparatus during cell replication and
mitosis. Dolastatin 10 (8), a linear peptide was first isolated from the sea-hare
Dolabella auricularia (Pettit et al., 1987) and subsequently re-isolated from the
marine cyanobacterium Symploca sp. confirming that the true source is the
cyanobacterium and not the sea-hare (Luesch et al., 2001 a). Dolastatin 10 binds to
tubulin and inhibits microtubule assembly and subsequently blocks cytokinesis. It has
also shown to be a non-competitive inhibitor of Vinca alkaloids (Bai et al., 1990) and
is currently in Phase II clinical trials for the treatment of renal carcinoma, lymphoma
and lymphocytic leukemia (Newman and Cragg, 2004a).
A Caribbean strain of L. majuscula was the source of curacin A (14), a
molecule that consists of an interesting thiazoline ring containing a 14-carbon alkyl
chain with conjugated diene and terminal olefin, and a methyl substituted cyclopropyl
moiety (Gerwick et al., 1994). Curacin A was shown to be selective for colon, renal
and breast cancer cell-lines when tested in NCI' s 60 cell line assay. It was later found
to be a potent inducer of tubulin depolymerization (1050 = 4.0 pM). Further testing
revealed that curacin A binds to the colchicine site of tubulin and stimulates the
uncoupled GTPase reaction that is typical of such agents. A series of semi-synthetic
analogs have been produced and curacin A is currently in preclinical trials as an
antiproliferative agent (Table 1.1) (Wipf et al., 2004). The curacin A biosynthetic gene
cluster has been recently characterized (Chang et al., 2004).
Cryptophycin A (20), a cyclic peptide was initially isolated from a terrestrial
cyanobacterium Nostoc sp. for its antifungal activity (Golakoti et al., 1994). The
compound exhibited strong cytotoxicity against KB (1050= 5 pglmL). Cryptophycin A
was found to be an effective inhibitor of tubulin polymerization, causing aggregation
of tubulin and depolymerization to linear polymers in a manner similar to Vinca
12
alkaloids and also inhibits vinbiastine binding to tubulin but not coichicine binding
suggesting that their binding sites may be identical or overlapping (Kerksiek et al.,
1995). Interestingly, a similar cyclic peptide, arenastatin A (21) was then isolated
from the Okinawan sponge Dysidea arenaria (Kobayashi et al., 1994). Cryptophycin
and its synthetic derivatives were in Phase I clinical trials as anticancer agents but
were withdrawn in 2002 (Newman and Cragg, 2004a).
L:J
HNO LOCH3
oi
Cryptophycin A (20)
°
HI'J
O HNO
OCH3
Arenastatin A (21)
( Cryptophycin-24)
ii. Actin-binding compounds
The actin cytoskeleton is a dynamic network of filaments which plays an
important role in maintaining cell shape, motility and signal transduction (Fenteany
and Zhu, 2003). Lyngbyabellins A (22) and B (23) are Lyngbya majuscula-derived
lipopeptides (Luesch et al., 2000a; Luesch et al., 2000b; Milligan et al., 2000) that are
structural homologs of dolabellin (24) isolated from the sea-hare, Dolabella
auricularia (Sone et al., 1995). Lyngbyabellin A (22) exhibited
IC50
values of 0.03
tg/mL and 0.50 ig/mL against KB cells (human nasopharyngeal carcinoma cell line)
and LoVo cells (human colon adenocarcinoma cell line), respectively. Lyngbyabellin
A was also shown to disrupt the microfilament network in fibroblastic A- 10 cells at
concentrations between 0.0 1-5.0 tg/mL. However, when treated with a higher
concentration of lyngbyabellin A many cells became binucleate, an observation which
is consistent for compounds inhibiting cytokinesis. Lyngbyabellin B (23) was found
to be less toxic than lyngbyabellin A (1050 values of 0.10 pg!mL and 0.83 pg/mL
13
against KB and LoVo cell lines, respectively) but exerted similar cellular effects as the
latter (Luesch et al., 2000a; Luesch et al., 2000b).
CI
CI
N=y
HNO
-m
S
o
0HNQ
0NJJ
N
-,
Lyngbyabellin A (22)
N
Lyngbyabellin B (23)
CI
Th,,O
NOH
OH
0
DCH3
Dolabellin (24)
iiii. Neurotoxic compounds
Antillatoxin (25), a cyclic lipopeptide was first reported as a potent
ichthyotoxin (Orj ala et aL, 1995). It is the most potent cyanobacterial ichthyotoxin to
date with with an LD50
0.05 ug/mL. Initial pharmacological studies showed that
antillatoxin was acutely neurotoxic and rapidly induced morphological changes in rat
cerebellar granule neurons (CGC's), including blebbing of neurite membranes
(Berman et al., 1999). Further testing showed that antillatoxin was a powerful
activator of voltage-gated sodium channels and mediated this activity through a unique
binding site (Li et al., 2001).
14
Antillatoxin (25)
Kalkitoxin (26), a linear lipopeptide was first isolated from a Caribbean
collection of L. majuscula and exhibited potent brine shrimp and fish toxicity (Wu,
1996). It was subsequently re-isolated in very small quantities from various Caribbean
collections of L. majuscula (Wu et al., 2000). Kalkitoxin was toxic to rat CGC's and
had an LC50 =3.86 nM (Berman et al., 1999) and there is also evidence indicating that
kalkitoxin is a blocker of voltage-sensitive Na channel in mouse neuro-2a cells (EC50
= 1 nM) (Wu et al., 2000).
Kalkitoxin (26)
The jamaicamides A-C (27-29) were isolated as highly functionalized
lipopeptides from a Jamaican strain of L. majuscula. Jamaicamaide A (27) was
particularly unique in its presence of an acetylenic bromide functionality. The
jamaicamides exhibited cytotoxicity against H-460 human lung cell line and mouse
neuro-2A neuroblastoma cell lines with an LC50
15 pM for both cell lines and
showed sodium channel blocking activity at a concentration of 5 tM (Edwards et al.,
2004). The recently characterized jamaicamide A biosynthetic gene cluster has
provided several powerful insights into the biosynthesis of this metabolite (Edwards et
al., 2004).
15
OCH3
NO
Jamaicamide A (27) R=Br
Jamaicamide B (28) R=H
OCH
'NO
Jamaicaniide C (29)
iv. Other interesting metabolites
Lyngbyatoxin A (30), was isolated from a Hawaiian shallow-water variety of
L. majuscula and is believed to be responsible for a condition known as 'swimmer's
itch' (Cardellina et al., 1979). This highly inflammatory compound had an
LD100 =
0.3 mg/kg in mice which was comparable to the toxicity of the teleocidins (Takashima
and Sakai, 1960). It was also found to be ichthyotoxic and exposure to 0.15 tg/mL
resulted in death of fish in 30 minutes (Cardellina et al., 1979). Ensuing
pharmacological studies showed that lyngbyatoxin A (30) was a potent tumor
promoter in a maimer similar to that of the phorbol esters when tested in mice (Fujiki
et al., 1984). The lynbyatoxin A biosynthetic gene cluster has been characterized from
a Hawaiian strain of L. majuscula (Edwards et al., 2004).
Lyngbyatoxin A (30)
Debromoaplysiatoxin (31)
Iri
Debromoaplysiatoxin (31), another highly inflammatory compound, was
isolated from an Okinawan collection of L. majuscula (Mynderse et al., 1977).
Further testing showed that the purified toxin induces cutaneous inflammation and
pustular folliculitis in humans and caused a severe inflammation in rabbits and hairless
mice upon topical application (Solomon and Stoughton, 1978).
L. bouillonii
isolated from Apra Bay, Guam, initially mis-identified as L.
majuscula, was found to produce a very potent cytotoxin, apratoxin A (32) which
exhibited an
IC50
= 0.52 nM and 0.36 nM against KB and LoVo cells, respectively.
However, its exact mode of action at present is uncharacterized (Luesch et al., 2001b).
A total synthesis of aparatoxin A has been reported (Chen and Forsyth, 2004) and the
synthesis of several structural analogs may provide insights into its mode of action.
OCH3
N
0
(
Apratoxin A (32)
The yellow-brown pigment, scytonemin (33), accumulates in the extracellular
sheath of many cyanobacteria (Nageli, 1849) but its structural determination was
completed only recently (Proteau et al., 1993). Structurally, the scytoneman skeleton
is very unique and is proposed to arise from the condensation of phenyipropanoid and
tryptophan units (Garcia-Pichel and Castenholz, 1991). It has since been established
that the production of scytonemin is a strategy to protect cells against the damaging
effects of ultraviolet radiation (UV) (Garcia-Pichel and Castenholz, 1991; Garcia-
17
Pichel et al., 1992). The production of scytonemin is induced only by UV and
increases proportionally to the flux received by the cell. This metabolite is also the
first described small molecule inhibitor of human polo-like kinase and is non-toxic,
making it an attractive candidate for the development of new anti-proliferatives
(Stevenson et al., 2002a; Stevenson et al., 2002b).
Scytonemin (33)
Marine cyanobacteria, especially Lyngbya
majuscula,
are clearly an
extraordinary source of complex and bio active secondary metabolites, and there is a
growing recognition that many of the more interesting natural products previously
attributed to marine invertebrates are indeed of cyanobacterial origin. The continued
discovery of new compounds from these metabolically talented organisms will
provide a range of novel and bioactive natural products which maybe developed as
potential therapeutics.
Nonribosomal peptide synthetase and polyketide synthases
A predominant theme encountered in marine cyanobacterial metabolites is the
rich integration of nonribosomal peptide synthetases (NRPS) and polyketide synthases
(PKS) to generate a seemingly endless number of novel metabolites (Gerwick et al.,
2001). PKS and NRPS are large multifunctional enzymes that share striking
organizational similarities. These enzymes have a modular arrangement and specific
domains within these modules are responsible for the activation, incorporation and
modification of simple carboxylic acids (PKS) or amino acid (NRPS) precursors
(Walsh, 2004). Each domain consists of highly conserved amino acid residues or core
motifs that enable the prediction or verification of domain function. The extensive
modification of these precursors or incorporation of unusual building blocks results in
unparalleled structural diversity (Cane et al., 1998; Walsh, 2004). A brief overview of
both biosynthetic systems is presented here.
Nonribosomalpeptide synthetases
Nonribosomal peptide biosynthesis is widespread in several bacteria and fungi
and plays an important role in the production of several biologically relevant products
(Schwarzer et al., 2003). These include antibiotics such as gramicidin S (34)
(Kratzschmar et al., 1989; Hon et al., 1989), bacitracin (35) (Gaidenko and
Khaikinson, 1988), and the penicillin precursor ACV-tripeptide (36) (Maccabe et al.,
1991); immunosuppressive agents such as cyclosporine (37) (Weber et al., 1994); and
siderophores such as enterobactin (38) (Liu et al., 1989) and myxochelin A (39)
(Silakowski et al., 2000).
NH2
NH
o
NH2
HN
0
SH
ACV-Tripeptide (36)
o
Gramicidin (34)
o
NHNH
o
NH
s
o
0
HO
0
Bacitracin :35)
HN
NH2 ON
NH
o,_N)i0H
HO
L)
19
hNCyclosporin (37)
HOi
0
HN
ONH
0
0
)OH
0
HNONH
OH
0
OH
0 QOH
OH
HON)OH
Moche1in A (39)
OH
OH
Enterobactin (38)
Three domains, the adenylation (A), peptidyl carrier protein (PCP) and
condensation (C) domain are involved in substrate recognition and activation, its
transfer to various catalytic centers and formation of peptide bond, respectively.
These three domains are integral parts of all NRPS modules and are often referred to
as the minimum module (Marahiel et al., 1997) (Figure 1.1). Nonribosomal peptides
are structurally more diverse than those that are ribosomally synthesized and this is
achieved by the incorporation of proteinogenic amino acids as well as "unusual"
amino acids such as a-hydroxy acids and carboxylic acids that may undergo further
modification such as N-, C-, or 0-methylation, heterocyclization, epimerization and
glycosylations mediated by associated tailoring enzymes (Finking and Marahiel, 2004;
Sieber and Marahiel, 2005).
20
1'
Condensation
Cl C2
Adenviation
Al A2 A3 A4 A5
ep tidvl carrier
teim
A6A7A8A9 AlO
Thioesterase
E
Required
I\
ZIZ2C3 Z3Z4Z5Z6Z7
Heterocyclization
MI M2 M3
N-Methylation
El E2 E3E4E5E6E7
Rl R2 R3R4R5R6 R7
Epimerization
Reductase
Optional
domains
Figure I.!: NRPS module organization. A minimum module is composed of
the three required domains, adenylation, peptidyl carrier protein and
condensation. Conserved amino acid residues within each domain are
represented as bold lines (Marahiel et al., 1997).
The adenylation domain (550 amino acids in size) is involved in the selection
of the cognate amino (carboxy) acid substrate and its activation as the aminoacyl
adenylate in a manner akin to t-RNA synthetases. But despite their catalytic similarity
to t-RNA synthetases, crystal structure of the phenylalanine activating A-domain from
gramicidin S synthetase revealed that its tertiary structure was more similar to firefly
luciferases (Conti et al., 1997). The crystal structure of this representative A- domain
along with sequence comparisons with other A-domains revealed that conserved
amino acid residues, most of which lie between the cores A4 and AS, played an
important role in determining substrate specificity (Stachelhaus et al., 1999).
Furthermore, these signature amino acid residues, referred to "codons", enable reliable
prediction of substrate specificity of newly identified A-domains (Figure I.2A)
(Challis et al., 2000; Stachelhaus et al., 1999). Selection of the cognate amino acid is
then followed by its activation as a highly reactive aminoacyl adenylate in a reaction
that requires ATP and Mg2 (Figure I.2B) (Finking and Marahiel, 2004).
21
A
NH2
NH
B
R
0
0
R
0
A
H2N(O + 0P0P0P0
H
I
0
O
Mg2
II
H
I
I
0-
Mg2
0-
PPi
OH OH
9
H2N
0
__kOri
OH OH
Figure 1.2: Recognition of cognate amino acid by binding pocket
residues (A) [figure adapted from (Challis et al.,2000)] and activation of amino
acid by the NRPS A-domain (B).
In the next step, the activated amino acid is transferred to the post-
translationally modified PCP domain. This modification involves the transfer of a
phosphopantetheinyl co-factor (Ppant) onto a conserved serine residue in the PCP
domain by a 4'-phosphopantetheinyl transferase (PPTase) to convert them to their
functionally active holo-form (Figure 1.3) (Finking and Marahiel, 2004). The Ppant
co-factor acts as a flexible arm and facilitates the transfer of the bound aminoacyl and
peptidyl substrates between various catalytic domains. Activated amino acids are
covalently tethered onto to the thiol group of the Ppant co-factor (Figure 1.4)
(Schwarzer et al., 2003).
22
NH2
HS'
H
N
H
N
-.
0
0
0E-0
HS'
H
H
N
N
0
OH
0
OH 4-PPtase
0
holo-PCP
apo-PCP
Figure 1.3: Post-translational modification of the PCP domain. Conversion of apoPCP to holo-PCP is achieved through the action of a dedicated 4'phosphopanteteinyl transferase which transfers a phosphopanteteinyl co-factor
R
R
H2N(AMP
£
.
-AMP
HN
Figure 1.4: Loading of PCP domain with activated amino acid
(Schwarzer et al., 2003).
The condensation domain of NRPS's (450 amino acids) is responsible for
peptide bond formation and chain translocation along the assembly line.
Condensation domains are localized between each consecutive A-PCP domains and
catalyze peptide bond formation between the electrophilic upstream peptidyl-S-PCP
and the free amine group of the downstream aminoacyl-S-PCP (Figure 1.5).
Additionally, studies have also revealed that the condensation domains may have an
editing function and are able to discriminate against amino acids of opposite
stereochemistry or larger side chains (Beishaw et al., 1999; Ehmann et al., 2000).
23
H
0
RNH 0
d
a
Ri
R2
SH
SH
Figure 15: Condensation domains catalyze peptide bond formation. PCP
domains of adjacent modules are loaded with activated cognate amino acids. The
electrophilic acceptor amino acid enters the donor site (d) and the nucleophilic
donor amino acid is located on the acceptor site (a) of the C-domain. After peptide
bond formation, the resulting dipeptide is transferred to donor site of the adjacent
C-domain. Figure adapted from (Finking and Marahiel, 2004).
Termination of nonribosomal peptide synthesis is catalyzed by a thioesterase
domain (TE) located at the C-terminal end of the last module (Finking and Marahiel,
2004). Chain release is occurs when the nascent peptidyl chain is transferred to an
active site serine present in the TE domain to generate an acyl-O-TE-enzyme
intermediate which is then attacked by a peptide nucleophile or water to form a
macrocycle or linear peptide product, respectively (Kohli and Walsh, 2003; Sieber and
Marahiel, 2003). Although macrocyclic release appears to be the preferred
mechanism, the mode of termination varies significantly between systems (Kohli et
al., 2001; Sieber and Marahiel, 2005).
Module 1
Module 2
Module 3
Module 1
Module 3
Module 2
Priming
R1
Cyclization/
Release
0
HN
LH
LH
Loading
0
R3
Module 1
Module 3
Module 2
Mode1Module2Module3
S
H
1-0
s
SH
H2NK
H2NK.
R1N \
R1
NH
H2N
R2
H
8
R3
Initiation
H
Termination
/1
H2
3
Elongation
S
R1<
H
NH
0=<
H2N
Figure 1.6: Steps involved in nonribosomal peptide biosynthesis. Figure adapted
from (Schwarzer et al., 2003)
In addition to the required domains involved in building the peptide backbone,
structural diversity is also introduced by the modification of the growing peptide chain
and is catalyzed by a variety of embedded tailoring domains such as epimerization (E),
N-methylation (N-Mt), oxidation (Ox), reduction (R). These auxiliary domains can
act in cis or in trans during the biosynthetic process (Finking and Marahiel, 2004).
The reactions of some of these tailoring domains are summarized in Figure 1.7.
25
Epimerization
domain
N-methyl transferase
domain
R
R
R
+H3N(
+H3Nri
R
H3N(
0
Heterocyclization
domain
S-adenosyl
methonine
(SAM)
o
HN
0
CH3
S
R
..,,.
+H3N(
+H3N
-2H0
OãHS
Oxidase
domain
R
Reductase
domain
0
0
-
+H3N
R
FMN
HJL FL j
N
+H3N
..7.__&:)L..ir0
R
H*L
R2
N
H
+NADPH
0
R
£
Figure 1.7: Reactions catalyzed by NRPS tailoring domains. Figure adapted from
(Schwarzer et al., 2003).
Polyketide synthases
Polyketides represent another important group of structurally diverse natural
products and include important antibiotics such as erythromycin A (40) (Cortes et al.,
1990; Donadio et al., 1991), immunosuppressive agents such as rapamycin (41)
(Schwecke et aL, 1995) and FK506 (42) (Motamedi et al., 1997) and antiparasitics
such as avermectin (43) (MacNeil et al., 1992). Polyketide synthases (PKS) are
multifunctional enzymes that catalyze the formation of complex polyketides by
repetitive addition of extender units derived from malonate or methyl malonate to an
activated carboxylic acid starter unit in a manner that is similar to fatty acid synthases
(Weissman, 2004). On the basis of their structure and biochemistry, three different
types of polyketide synthases have been described (Staunton and Weissman, 2001).
The type I PKS ' s are modular enzymes similar to NRPS 's in architecture and consist
of distinct domains that catalyze incorporation of simple precursors. The type I PKS
involved in the biosynthesis of 6-deoxyerythronlide B, the non-glycosylated precursor
of erythromycin A (40) is one of the best examples of such systems (Khosla, 1997;
McDaniel et al., 2005). A brief overview of modular type I PKS's is presented in the
following sections. In contrast, type II PKS's are composed of iteratively acting
individual proteins that generate poly-3-keto intermediates and associated
ketoreductases, cyclases and aromatases catalyze the formation of multicyclic
aromatics like actinorhodin (44) (Staunton and Weissman, 2001). The type III PKS's
in bacteria are a newly characterized group and are similar to chalcone synthases of
higher plants. They are distinct from type I and type II PKS '5 in that they use free
CoA thioesters as substrates without the involvement of 4'-phosphopantheteine co-
factor (Moore and Hopke, 2001; Moore et al., 2002). The recently characterized
enterocin (45) biosynthetic gene cluster from Streptomyces maritimus is a good
example of a type III polyketide synthase-derived product (Piel et al., 2000).
27
OMe
Erythromycin A (40)
Rapamycin (41)
_
FK506 (42)
Avermectjn Bib (4
HO
I
Ii
OH 0
COOH
Actinorhodjn (44)
Enterocjn (45)
Modular type I PKS's are architecturally similar to NRPS's and possess
mandatory or core domains along with additional tailoring domains. In the case of
PKS, a minimum module is composed of the ketosynthase (KS), acyl transferase (AT),
acyl carrier protein (ACP) and thioesterase domains. A variable number of ancillary
domains such as the ketoreductase domain, enoyl reductase domain and dehydratase
domains are also associated with PKS's (Cane and Walsh, 1999; Staunton and
Weissman, 2001).
Carrier Protein
Thioeste:::
Optional domains
Figure 1.8: PKS module organization. The ketosynthase, acyl transferase and
acyl carrier protein are the required domains and compose the minimum module.
Additionally, auxiliary domains such as the ketoreductase, enoyl reductase and
dehydratase may also be present.
The first step in polyketide biosynthesis is mediated by the AT domain which
transfers activated precursors to the corresponding modified ACP domain and is
similar to NRPS A-domains in this regard (Figure 1.9). However, while A-domains
also catalyze the activation of amino acid substrates the AT domains receive substrates
in the activated form (malonyl-CoA or methylmalonyl-CoA). It has been established
that substrate specificity of the AT-domain for malonyl-CoA or methylmalonyl-CoA
is determined by the presence of certain conserved amino acid residues (Haydock Ct
al., 1995). Modification of the ACP domain involves the posttranslational attachment
of the phospantetheinyl co-factor onto a conserved serine residue in a manner similar
to NRPS PCP domains. Although they have similar functions, ACP and PCP domains
29
do not reveal any obvious sequence similarities except for the conserved serine residue
(Finking and Marahiel, 2004).
0
0
0
0
-OS-CoA i
R
R
Figure 1.9: Recognition of substrate by the AT domain and its transfer to ACP
domain.
The KS domain mediates the fundamental chain elongation reaction and
catalyzes C-C bond formation through a Claisen-type condensation. The KS domain
first decarboxylates the malonyl or methylmalonyl extender unit bound to the ACP
domain. The resulting nucleophile then reacts with the thio ester group of the
polyketide chain attached to the KS domain and ultimately this decarboxylative
condensation results in the extension of the growing ketide chain by two carbons
(Figure 1.10) (Katz, 1997).
R)(L
L
Figure 1.10: Ketide bond formation catalyzed by the KS domain.
Ii]
The ketoreductase domain (KR), dehydratase domain (DH) and the enoyl
reductase domain (ER) are auxiliary domains that catalyze three consecutive reductive
reactions. Before transfer of the elongated polyketide chain to the KS domain of the
adjoining module, the 13-keto group can be reduced to a hydroxyl by the KR domain
and requires NADPH as a co-factor. The dehydratase domain catalyzes the
dehydration of the J3-hydroxyl group resulting in the formation of a double bond.
Finally, the enoyl reductase uses an NADPH co-factor to form a saturated bond in the
ketide chain (Figure 1.11) (Katz, 1997). After the final extension, the nascent ketide
chain is transferred to the TE domain which is responsible for product release and
cyclization.
KR
OH
0
RJUJLNHRJ1JL
OH
ER
RJ1
R'
Figure 1.11: Reactions catalyzed by PKS auxiliary domains. The ketoreductase domain
(KR) catalyzes reduction of the 13-keto function, the dehydratase domain (DH) mediates
dehydration of the -hydroxyl function and further reduction to form a saturated bond is
catalyzed by the enoyl reductase domain (ER).
Although modular type I PKS's and NRPS's employ a similar biosynthetic
logic, they exhibit significant differences in quaternary structure. In the double-helical
model proposed for modular type I PKS's, each subunit consists of identical
polypeptides twisted around each other in a helix. At the core of the helix are the KS,
AT and ACP domains, while the reductive domains are accommodated at the
periphery (Staunton et al., 1996). On the other hand, NRPS's are monomeric (Sieber
et al., 2002) and in hybrid systems it is believed that the dimeric PKS forms a core,
with NRPS loops (Weissman, 2004). Hybrid PKS/NRPS clusters combine both
31
strategies for the production of structurally complex and therapeutically active
molecules such as epothiolone (46) from Sorangium cellulosum (Molnar et al., 2000),
myxothiazol (47) from Stigmatella auriantica DW 4/3-1 (Silakowski et al., 1999),
yersiniabactin (48) from Yersinia pestis (Gehring et al., 1998), and bleomycin (49)
from Streptomyces verticillus (Du et aL, 2000).
NH
Myxothiazol (47)
Epothiolone (46)
H2NO
OH
S
H
H2
H
OH
Xl'oH
NN
0
H
0
ii
HN,
Yersiniabactin (48)
OH
\\
I
N
/
H
Bleomycin A2 (49)
ni-i
OH
The modularity of these natural product assembly lines and permissivity of
tailoring enzymes offer exciting prospects for the generation of entirely new or
modified products with optimal therapeutic potential through combinatorial
biosynthesis (Cane et al., 1998; Walsh, 2004). Despite several daunting challenges,
steady progress is being made in the field of combinatorial biosynthesis (McDaniel et
al., 2005; Sieber and Marahiel, 2005). Advances have been made in understanding
crucial interactions between different modules in PKS and NRPS systems (Hahn and
32
Stacheihaus, 2004; Wu et al., 2001). Several PKS/NRPS gene clusters such as those
encoding DEBS and yersniabactin have been successfully expressed in E. coil (Pfeifer
and Khosla, 2001; Pfeifer et al., 2001; Pfeifer et al., 2003). Similarly, the production
of artificial nonribosomal peptide products in an engineered E. coil host has also been
reported (Gruenewald et al., 2004). Recent work to exploit the chemoenzymatic
potential of TE domains has also demonstrated its ability to catalyze the robust
cyclization of linear peptide and mixed peptide/polyketide metabolites to their final
macrocyclic forms (Trauger et al., 2001; Kohli et al., 2002b; Kohli et al., 2002a;
Watanabe et al., 2003; Boddy et al., 2003; Sieber et al., 2003). Another strategy by
which biosynthetic pathways can be utilized to generate natural products or natural
product-like compounds is by partial pathway expression and precursor directed
biosynthesis. For example, in the case of the epothilone biosynthesis from the
myxobacterium Sorangium cellulosum, an
E.
coil strain was engineered to express the
last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF)
and the substrate required to complement the biosynthetic enzymes was obtained by
chemical synthesis. Under high-density cell culture conditions, this engineered E.
coil
strain processed exogenously fed synthetic substrate into epothilone C (Boddy et al.,
2004).
Secondary metabolite biosynthetic gene clusters from marine cyanobacteria
The rich integration of PKS/NRPS systems is the predominant biogenetic
theme in marine cyanobacterial metabolites. Although these enzymes are similar to
those present in terrestrial organisms, many of the metabolites from marine organisms
possess unique functional groups that are not encountered in the former. The unique
structural features and bio active properties of many cyanobacterial metabolites have
prompted efforts to study the biosynthesis of these compounds at the molecular
genetics level. The overarching goal of current research in this area is to functionally
express cyanobacterial secondary metabolite gene clusters in a heterologous host. Not
only will such technologies enable increased production of compounds of biomedical
importance, but also will aid in the development of strategies aimed at combinatorial
33
biosynthesis of these metabolites. Analysis of structural types presented in Table 1.1
indicates that a majority of these compounds are derived from modular PKSINRPS's
(Salomon et al., 2004). Additionally, there is growing recognition that microbial
symbionts are often important contributors to the biosynthesis of biologically active
compounds (Piel, 2004). Therefore, knowledge gleaned from in-depth analysis of
these gene clusters can led to the development of genetic systems capable of
overexpressing these metabolites in order to provide an unlimited supply for clinical
evaluation, thus circumventing the supply issue.
The increasing inventory of PKS/NRPS biosynthetic gene clusters available in
public databases has facilitated the use of these sequences to design targeted cloning
strategies to isolate new biosynthetic gene clusters from marine organisms.
Additionally, the isolation and characterization of several freshwater and terrestrial
cyanobacterial gene clusters such as those involved in the biosynthesis of microcystin
(18) (Tillett et aL, 2000), nodularin (19) (Moffitt and Neilan, 2004), nostopeptolides
(50) (Hoffmann et al., 2003), nostocyclopeptide (51) (Becker et al., 2004) and
anabaenopeptilide (52) (Rouhiainen et al., 2000) have provided the foundation for
further pursuit of similar gene clusters from their marine counterparts.
CH3
H
00
0
HO'
0
ONH
HNO
O
0
ONH2
H
Nostopeptolide A (50)
HN
0
H
HN°
H0Ny
ss"
0
Nostocyclopeptide Al (51)
34
OH
HO
OCH3CCI3
HN
1y4Ny=&kH
LJ N"S
1
Barbamide (53)
0NH
-
OH
CI
rAnabaenopeptilide
90B (52)
Inherent difficulties of culturing marine organisms, such as cyanobacteria and
the lack of effective techniques to genetically manipulate these organisms have
hindered research efforts in this area. However, the ability to culture certain marine
cyanobacteria like Lyngbya majuscula along with improved methods to isolate DNA
and generate genomic libraries has made it possible to initiate molecular genetic
studies with these organisms. Only recently have the genetic architectures of several
cyanobacterial biosynthetic gene clusters been determined, and studies to understand
and exploit this biosynthetic machinery present an exciting new area of research. At
present, four secondary metabolite biosynthetic gene clusters have been described in
the literature from different strains of L. majuscula.
The barbamide (53) biosynthetic gene cluster (bar) was the first marine
cyanobacterial gene cluster to be elucidated (Chang et al., 2002). It is a mixed
PKS/NRPS system with unusual features that include a stand-alone peptidyl carrier
(PCP) and unusual A-domains, as well as a PKS module that is encoded on two
separate ORFs. The biosynthetic system also encodes several tailoring enzymes
involved in the chlorination, a-oxidation, and decarboxylation of leucine to form a
trichioroisovaleric acid moiety, and the oxidative decarboxylation of the cysteine
residue at the end of the assembly line to form the terminal thiazole ring (Figure 1.12)
(Chang et al., 2002).
35
PCP
Put.
haIogenase
L-amino
PKS
NRPS NRPS-PKS
Decarboxylase oxidase
NRPS
barBil
barA
B2 barC
barD
barE
barG
barF
barH
bar/-K
D DDDE'EDEF_______________
4
26kb
Figure 1.12: The barbamide (53) biosynthetic gene cluster.
In addition to the barbamide biosynthesic gene cluster, the curacin A (14)
biosynthetic gene cluster was also isolated from this same strain of L. majuscula
(Chang et al., 2004). A particularly notable feature of the pathway involves a unique
biochemical mechanism for cyclopropyl ring formation mediated via a novel enzyme
that bears homology with 3-hydroxy-3 -methyl glutaryl CoA (HMG-CoA) synthases
(Figure 1.13) (Chang et al., 2004).
PKS
curA
I
HMG
NRPS
cassette
- Pics
PKS
PKS
PKS
PKS
PKS
curB-E
curF
curG
curH
curl
curJ
curK
>DDDD1
X
)_____
)I
PKS TE Hyd
PKS
curL
l
curM
l
N
___)D
64kb
Figure 1.13: The curacin A (14) biosynthetic gene cluster.
The 58 kb jamaicamide A (27) biosynthetic gene cluster (jam) is a remarkable
example of a co-linear pathway for the assembly of a complex lipopeptide and
represents a highly integrated mixed PKS/NRPS pathway (Figure 1.14) (Edwards et
aL, 2004). The jamaicamide pathway possesses a range of genetic elements that
encode for enzymes catalyzing several biochemical transformations including a novel
alkynyl PKS starter unit, an HMG-CoA synthase containing gene cassette for pendent
vinyl or vinyl chloride formation, incorporation of a decarboxylase domain into JamL
NRPS module, and chain termination resulting in pyrrolinone ring formation.
HMC
jamA-D
PKS
cassette
famE
jamF-I
DDDDI
PKS-
PKS
PKS
jamJ
jamK
NRPS
jamL
DE
DDDiX
M
PKS tIRPS PKS Con
N
P
0
Q
XE
a
58kb
Figure 1.14: The jamaicamide A (27) biosynthetic gene cluster.
The lyngbyatoxin biosynthetic gene cluster was cloned and characterized from
a Hawaiian strain of L. majuscula (Edwards and Gerwick, 2004). The
11 kb cluster
consists of two NRPS modules that catalyze the formation the L-N-Me-Val-Ltryptophanol which is modified to form the indolactam-V by a P450 monooxygenase.
A prenyl transferase then catalyzes the attachment of the geranyl group to form
lyngbyatoxin A (Figure 1.15) (30).
P450
NRPS
monooxygenase
ItxA
ItxB
Prenyl
transferase
ItxC
Reductasel
Oxidase
ItxD
11.3 kb
Figure 1.15: The lyngbyatoxin A (30) biosynthetic gene cluster.
The study of marine cyanobacterial natural product chemistry has revealed a
number of metabolic themes as well as novel and re-occurring structural motifs. An
overriding theme is the rich integration of polyketide/peptide units. Other common
themes include the incorporation of novel starter units (e.g. tertiary-butyl, terminal
alkene, alkynes, and even a bromo-alkyne in 27), a number of diverse halogenations,
heterocylces, the incorporation of functionalized pendent carbons from the action of
37
an HMGCoA synthase gene cassette, and the incorporation of diverse amino acid units
(e.g. j3-amino acids, N-methylated and 0-methylated amino acids). The genetic
features that encode for these functional units in marine cyanobacteria will provide a
rich toolbox for the creation of structural diversity through pathway engineering and
chemoenzymatic synthesis.
GENERAL THESIS CONTENTS
Marine cyanobacteria are an extraordinary source of complex and bioactive
secondary metabolites that are derived from hybrid PKS/NRPS systems. Along with
the search for new and bioactive metabolites from marine cyanobacteria, our
laboratory has also undertaken studies to understand the biosynthesis of these
metabolites at the molecular genetic level. A Jamaican strain of the cyanobacterium L.
majuscula was the source of the novel cytotoxic and antifungal metabolite,
hectochiorin. The unique biological properties and presence of intriguing structural
features in hectochiorin, such as the gem dichioro group and two 2,3-dihydroxy
isovaleric acid units, prompted us to clone and characterize its biosynthetic gene
cluster to obtain details regarding its biosynthesis at the molecular genetic level.
Initial attempts to isolate the hectochiorin biosynthetic gene cluster led to the
serendipitous discovery of a cryptic gene cluster from the genome of the organism.
Details regarding the cloning, sequence characterization and demonstration of this
cryptic cluster expression are reported in chapter two. A novel interdisciplinary
approach designed to identify the metabolite produced by this cryptic cluster is also
discussed.
The isolation, sequence characterization and partial biochemical
characterization of the hectochlorin biosynthetic gene cluster is discussed in chapter
three. The 38 kb gene cluster possesses several unusual features such as the presence
of KR domains in two peptide synthetase modules which are predicted to be involved
in the formation of the two 2,3-dihydroxy isovaleric acid (DHiv) units and appears to
be the first report of such an arrangement in a cyanobacterial secondary metabolite
gene cluster. Also present are two cytochrome P450 monooxygenases and a putative
halogenase.
The gene cluster encoding for the biosynthesis of carmabin was elucidated
from a Caribbean L. majuscula (Z. Chang, D. Sherman, W.H. Gerwick, unpublished)
and chapter four provides details regarding the expression and biochemical
characterization of two adenylation domains from this gene cluster. The concluding
chapter will provide a brief summary and perspective of the preceding chapters.
39
REFERENCES
Aicher, T. D., Buszek, K. R., Fang, F. G., Forsyth, C. J., Jung, 5. 0., Kishi, Y.,
Matelich, M. C., Scola, P. M., Spero, D. M., and Yoon, S. K. (1992). Total synthesis
of halichondrin B and norhalichondrin B. Journal of American Chemical Society 114,
3 162-3 164.
Amador, M. L., Jimeno J., Paz-Ares L., Cortes-Funes H., and Hidago M. (2003).
Progress in the developement and acquisition of anticancer agents from marine
sources. Annals of Oncology 14, 1607-1615.
Bai, R., Pettit, G. R., and Hamel, E. (1990). Dolastatin 10, a powerful cytostatic
peptide derived from a marine animal. Inhibition of tubulin polymerization mediated
through the ymca alkaloid binding domain. Biochemical Pharmacology 39, 19411949.
Barry,B.A., Boemer,R.J., and dePaula,J.C. (1994). The use of cyanobacteria in the
study of the structure and function of photosystem II. In The molecular biology of
cyanobacteria, D.A.Bryant, ed. (Netherlands: Kiuwer Academic Publishers), pp. 217257.
Batard, P., Szollosi, J., Luescher, I., Cerottini, J. C., MacDonald, R., and Romero, P.
(2002). Use of phycoerythrin and allophycocyanin for fluorescence resonance energy
transfer analyzed by flow cytometry: advantages and limitations. Cytometry 48, 97105.
Becker, J. E., Moore, R. E., and Moore, B. S. (2004). Cloning,sequencing,and
biochemical characterization of the nostocyclopeptide biosynthetic gene
cluster:molecular basis for imine macrocyclization. Gene 325, 35-42.
Beishaw, P. J., Walsh, C. T., and Stachelhaus, T. (1999). Aminoacyl-CoAs as probes
of condensation domain selectivity in nonribosomal peptide synthesis. Science 284,
486-489.
Bergman, W. and Feeney, R. J. (1951). Marine products. XXXII. The nucelosides of
sponges. I. Journal of Organic Chemistry 16, 98 1-987.
Berman, F. W., Gerwick, W. H., and Murray, T. F. (1999). Antillatoxin and
kalkitoxin, ichthyotoxins from the tropical cyanobacterium Lyngbya majuscula,
induce distinct temporal patterns of NMDA receptor-mediated neurotoxicity. Toxicon
37, 1645-8.
Blunt, J. W., Copp, B. R., Munro, M. H., Northcote, P. T., and Prinsep, M. R. (2005).
Marine natural products. Natural Product Reports 22, 15-61.
!EIJ
Boddy, C. N., Hotta, K., Tse, M. L., Watts, R. E., and Khosla, C. (2004). Precursordirected biosynthesis of epothilone in Escherichia coli. Journal of American Chemical
Society 126, 7436-7437.
Boddy, C. N., Schneider, T. L., Hotta, K., Walsh, C. T., and Khosla, C. (2003).
Epothilone C macrolactonization and hydrolysis are catalyzed by the isolated
thioesterase domain of epothilone polyketide synthase. Journal of American Chemical
Society 125, 3428-3429.
Briand, J. F., Jacquet, S., Bernard, C., and Humbert, J. F. (2003). Health hazards for
terrestrial vertebrates from toxic cyanobacteria in surface water ecosystems.
Veterinary Research 34, 36 1-377.
Burgoyne, D. L., Andersen, R. J., and Allen, T. M. (1992). Contignasterol, a highly
oxygenated steroid with the unnatural 14b configuration from the marine sponge
Petrosia contignata Thiele, 1899. Journal of Organic Chemistry 57, 525-528.
Burja, A. M., Banaigs, B., Abou-Mansour, E., Burgess, J. G., and Wright, P. C.
(2001). Marine cyanobacteria: A prolific source of natural products. Tetrahedron 57,
9347-9377.
Cane, D. E. and Walsh, C. T. (1999). The parallel and convergent universes of
polyketide synthases and nonribosomal peptide synthetases. Chemistry & Biology 6,
3 19-325.
Cane, D. E., Walsh, C. T., and Khosla, C. (1998). Harnessing the biosynthetic code:
combinations, permutations, and mutations. Science 282, 63-68.
Cardellina, J. H., Marner, F. J., and Moore, R. B. (1979). Seaweed dermatitis: structure
of lyngbyatoxin A. Science 204, 193-195.
Castenholz,R.W., Rippka,R., and Herdman,M. (2001). Bergey's Manual of Systematic
Bacteriology Volume I. (New York: Springer-Verlag).
Challis, G. L., Ravel, J., and Townsend, C. A. (2000). Predictive, structure-based
model of amino acid recognition by nonribosomal peptide synthetase adenylation
domains. Chemistry & Biology 7, 2 11-224.
Chang, Z., Flatt, P., Gerwick, W. H., Nguyen, V. A., Willis, C. L., and Sherman, D. H.
(2002). The barbamide biosynthetic gene cluster: a novel marine cyanobacterial
system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS)
origin involving an unusual trichloroleucyl starter unit. Gene 296, 23 5-247.
Chang, Z., Sitachitta, N., Rossi, J. V., Roberts, M. A., Flatt, P. M., Jia, J., Sherman, D.
H., and Gerwick, W. H. (2004). Biosynthetic pathway and gene cluster analysis of
41
curacin A, an antitubulin natural product from the tropical marine cyanobacterium
Lyngbya majuscula. Journal of Natural Products 67, 1356-1367.
Chen, J. and Forsyth, C. J. (2004). Total synthesis of the marine cyanobacterial
cyclodepsipeptide apratoxin A. Proc Nat! Acad Sci U S A 101, 12067-12072.
Conti, E., Stacheihaus, T., Marahiel, M. A., and Brick, P. (1997). Structural basis for
the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S.
Embo Journal 16, 4174-4183.
Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J., and Leadlay, P. F. (1990). An
unusually large multifunctional polypeptide in the erythromycin- producing polyketide
synthase of Saccharopolyspora erythraea. Nature 348, 176-8.
Coulson, F. R. and O'Donnell, S. R. (2000). The effects of contignasterol (JZP-94,005)
on allergen-induced plasma protein exudation in the tracheobronchial airways of
sensitized guinea-pigs in vivo. Inflammation Research 49, 123-127.
Donadio, S., Stayer, M. J., McAlpine, J. B., Swanson, S. J., and Katz, L. (1991).
Modular organization of genes required for complex polyketide biosynthesis. Science
252, 675-679.
Donia, M. and Hamarm, M. T. (2003). Marine natural products and their potential
applications as anti-infective agents. Lancet Infectious Diseases 3, 338-348.
Dougherty, T. J., Barrett, J. F., and Pucci, M. J. (2002). Microbial genomics and novel
antibiotic discovery: new technology to search for new drugs. Current Pharmaceutical
Design 8, 1119-1135.
Du, L., Sanchez, C., Chen, M., Edwards, D. 3., and Shen, B. (2000). The biosynthetic
gene cluster for the antitumor drug bleomycin from Streptomyces verticillus
ATCC 15003 supporting functional interactions between nonribosomal peptide
synthetases and a polyketide synthase. Chemistry & Biology 7, 623-642.
Edwards, D. 3. and Gerwick, W. H. (2004). Lyngbyatoxin biosynthesis: sequence of
biosynthetic gene cluster and identification of a novel aromatic prenyltransferase.
Journal of American Chemical Society 126, 11432-11433.
Edwards, D. J., Marquez, B. L., Nogle, L. M., McPhail, K., Goeger, D. E., Roberts, M.
A., and Gerwick, W. H. (2004). Structure and biosynthesis of the jamaicamides, new
mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya
majuscula. Chemistry & Biology]], 817-833.
Egner, P. A., Wang, J. B., Zhu, Y. R., Zhang, B. C., Wu, Y., Zhang, Q. N., Qian, G.
S., Kuang, S. Y., Gange, S. J., Jacobson, L. P., Helzlsouer, K. J., Bailey, G. S.,
Groopman, J. D., and Kensler, T. W. (2001). Chlorophyllin intervention reduces
42
aflatoxin-DNA adducts in individuals at high risk for liver cancer. Proceedings of the
National Academy of Sciences of the United States of America 98, 1460 1-14606.
Ehmann, D. E., Trauger, J. W., Stacheihaus, T., and Walsh, C. T. (2000). AminoacylSNACs as small-molecule substrates for the condensation domains of nonribosomal
peptide synthetases. Chemistry & Biology 7, 765-772.
Erba, E., Bergamaschi, D., Ronzoni, S., Faretta, M., Taverna, S., Bonfanti, M.,
Catapano, C. V., Faircloth, G., Jimeno, J., and D'Inalci, M. (1999). Mode of action of
thiocoraline, a natural marine compound with antitumor activity. Bristish Journal of
Cancer 80, 1901-1907.
Fehling, R. H., Buchanan, G. 0., Mincer, T. J., Kauffman, C. A., Jensen, P. R., and
Fenical, W. (2003). Salinosporamide A: a highly cytotoxic proteasome inhibitor from
a novel microbial source, a marine bacterium of the new genus Salinospora. Angew
Chem mt Ed Engl 42, 355-357.
Fenteany, G. and Zhu, 5. (2003). Small-molecule inhibitors of actin dynamics and cell
motility. Current Topics in Medicinal Chemistry 3, 593-6 16.
Ferber, M., Sporning, A., Jeserich, G., DeLaCruz, R., Watkins, M., Olivera, B. M.,
and Terlau, H. (2003). A novel conus peptide ligand for K+ channels. Journal of
Biological Chemistry 278, 2177-2183.
Finking, R. and Marahiel, M. (2004). Biosynthesis of nonribosomal peptides. Annual
Review of Microbiology 58, 453-488.
Fujiki, H., Suganuma, M., Hakii, H., Bartolini, G., Moore, R. E., Takayama, S., and
Sugimura, T. (1984). A two-stage mouse skin carcinogenesis study of lyngbyatoxin A.
Journal of Cancer Research and Clinical Oncology 108, 174-6.
Gaidenko, T. A. and Khaikinson, M. (1988). Cloning of the genes controlling the
biosynthesis of bacitracin in Bacillus lichenzformis. Molekuliarnaia genetika,
mikrobiologiia i virusologiia 2, 24-28.
Garcia-Pichel, F. and Castenholz, R. (1991). Characterization and biological
implications of scytonemin, a cyanobacterial sheath pigment. Journal of Phycology 27,
395-405.
Garcia-Pichel, F., Sherry, N. D., and Castenholz, R. (1992). Evidence for an
ultraviolet sunscreen role of the extracellular pigment scytonemin in the terrestrial
cyanobacterium, Chiorogleopsis sp. Photochemistry and Photobiology 56, 17-23.
Garcia-Rocha, M., Bonay, P., and Avila, J. (1996). The antitumoral compound
kahalalide F acts on cell lysosomes. Cancer Letters 99, 43-50.
43
Gehring, A. M., Mori, I., Perry, R. D., and Walsh, C. T. (1998). The nonribosomal
peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of
yersiniabactin, an iron-chelating virulence factor of Yersinia pestis. Biochemistry 37,
11637-11650.
Gerwick, W. H., Proteau, P. J., Nagle, D., Hamel, E., Blokhin, A., and Slate, D. L.
(1994). Structure of curacin A, a novel antimitotic, antiproliferative, and brine shrimp
toxic natural product from the marine cyanobacterium Lyngbya majuscula. Journal of
Organic Chemistry 59, 1243-1245.
Gerwick,W.H., Tan,L., and Sitachitta,N. (2001). Nitrogen-containing metabolites from
marine cyanobacteria. In The Alkaloids, G.A.Cordell, ed. (San Diego: Academic
Press), pp. 75-184.
Giovannoni, S. J., Turner, S., Olsen, G. J., Barns, S., Lane, D. J., and Pace, N. R.
(1988). Evolutionary relationships among cyanobacteria and green chloroplasts.
Journal of Bacteriology 170, 3 584-3592.
Golakoti, T., Ohtani, I., Patterson, G. M., Moore, R. E., Corbett, T. H., Valeriote, F.
A., and Demchik, L. (1994). Total Structures of Cryptophycins, Potent Antitumor
Depsipeptides from the Blue-Green Alga Nostoc sp. Strain GSV 224. Journal of
American Chemical Society 116, 4729-4737.
Grassle, J. F. and Maciolek, N. J. (1992). Deep-sea species richness: regional and local
diversity estimates from quantitative bottom samples. American Naturalist 139, 313341.
Gruenewald, S., Mootz, H. D., Stehmeier, P., and Stachelhaus, T. (2004). In vivo
production of artificial nonribosomal peptide products in the heterologous host
Escherichia coli. Applied and Environmental Microbiology 70, 3282-3291.
Gunasekera, S. P., Gunasekera, M., Longley, R. E., and Schulte, G. K. (1990).
Discodermolide: a new bioactive polyhydroxylated lactone from the marine sponge
Discodermia dissoluta. Journal of Organic Chemistry 55, 4912-4915.
Hahn, M. and Stacheihaus, T. (2004). Selective interaction between nonribosomal
peptide synthetases is facilitated by short communication-mediating domains.
Proceedings of the National Academy of Sciences of the United States of America
101, 15585-15590.
Hamann, M. T. and Scheuer, P. J. (1993). Kahalalide F: a bioactive depsipeptide from
the sacoglossan mollusk Elysia rufescens and the green alga Bryopsis sp. Journal of
American Chemical Society 115, 5825-5826.
Haydock, S. F., Aparicio, J. F., Molnar, I., Schwecke, T., Khaw, L. E., Konig, A.,
Marsden, A. F., Galloway, I. S., Staunton, J., and Leadlay, P. F. (1995). Divergent
sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl
carrier protein transacylase domains in modular polyketide synthases. Febs Letters
374, 246-248.
Hofffiiann, D., Hevel, J. M., Moore, R. E., and Moore, B. S. (2003). Sequence analysis
and biochemical characterization of the nostopeptolide A biosynthetic gene cluster
from Nostoc sp. GSV224. Gene 311, 171-180.
Hori, K., Yamomoto, Y., Minetoki, T., Kurotsu, T., Kanda, M., Miura, S., Okamura,
F., Furuyama, J., and Saito, Y. (1989). Molecular cloning and nucleotide sequence of
the gramicidin S synthetase 1 gene. Journal of Biochemistry (Tokyo) 106, 63 9-645.
Ishida, T., Watanabe, M. M., Sugiyama, J., and Yokota, A. (2001). Evidence for
polyphyletic origin of the members of the orders of Oscillatoriales and Pleurocapsales
as determined by their 16S rDNA analysis. FEMS Microbiology Letters 201, 79-82.
Iwasaki, H. and Kondo, T. (2004). Circadian timing mechanism in the prokaryotic
clock system of cyanobacteria. Journal of Biological Rhythms 19, 348-360.
Jensen, P. R., Mincer, T. J., Williams, P. G., and Fenical, W. (2005). Marine
actinomycete diversity and natural product discovery. Antonie Van Leeuwenhoek 87,
43-48.
Jimeno, J., Lopez-Martin, J. A., Ruiz-Casado, A., Izquierdo, M. A., Scheuer, P. J., and
Rinehart, K. (2004). Progress in the clinical development of new marine-derived
anticancer compounds. Anticancer Drugs 15, 321-329.
Kalesse, M. (2000). The chemistry and biology of discodermolides. Chembiochem 1,
171-175.
Katz, L. (1997). Manipulation of modular polyketide synthases. Chemical Reviews 97,
2557-2575.
Kerksiek, K., Mejillano, M. R., Schwartz, R. E., Georg, G. I., and Himes, R. H.
(1995). Interaction of cryptophycin 1 with tubulin and microtubules. Febs Letters 377,
59-61.
Khosla, C. (1997). Harnessing the biosynthetic potential of modular polyketide
synthases. Chemical Reviews 97, 2577-2590.
Kobayashi, M., Aoki, S., Ohyabu, N., Kurosum, M., Wang, W., and Kitagawa, I.
(1994). Arenastatin A, a potent cytotoxic depsipeptide from the Okinawan marine
sponge Dysidea arenaria. Tetrahedron Letters 35, 7969-7972.
45
Kobayashi, M., Natsume, T., Tamaoki, S., Watanabe, J., Asano, H., Mikami, T.,
Miyasaka, K., Miyazaki, K., and Gondo, M. (1997). Antitumor activity of TZT-1027,
a novel dolastatin 10 derivative. Japanese Journal of Cancer Research 88, 316-327.
Kohli, R. M., Takagi, J., and Walsh, C. T. (2002a). The thioesterase domain from a
nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides.
Proceedings of the National Academy of Sciences of the United States of America 99,
1247-1252.
Kohli, R. M., Trauger, J. W., Schwarzer, D., Marahiel, M. A., and Walsh, C. T.
(2001). Generality of peptide cyclization catalyzed by isolated thioesterase domains of
nonribosomal peptide synthetases. Biochemistry 40, 7099-7 108.
Kohli, R. M. and Walsh, C. T. (2003). Enzymology of acyl chain macrocyclization in
natural product biosynthesis. Chemical Communication (Camb) 7, 297-307.
Kohli, R. M., Walsh, C. T., and Burkart, M. D. (2002b). Biomimetic synthesis and
optimization of cyclic peptide antibiotics. Nature 418, 658-661.
Kratzschmar, J., Krause, M., and Marahiel, M. A. (1989). Gramicidin-S Biosynthesis
Operon Containing the Structural Genes Grsa and Grsb Has an Open Reading Frame
Encoding a Protein Homologous to Fatty Acid Thioesterases. Journal of Bacteriology
171, 5422-5429.
Li, W. I., Berman, F. W., Okino, T., Yokokawa, F., Shioiri, T., Gerwick, W. H., and
Murray, T. F. (2001). Antillatoxin is a marine cyanobacterial toxin that potently
activates voltage-gated sodium channels. Proceedings of the National Academy of
Sciences of the United States of America 98, 7599-7604.
Lindel, T., Jensen, P. R., Fenical, W., Long, B. H., Casazza, A.-M., Carboni, J., and
Fairchild, C. R. (1997). Eleutherobin, a New Cytotoxin that Mimics Paclitaxel (Taxol)
by Stabilizing Microtubules. Journal of American Chemical Society 119, 8744-8745.
Liu, J., Duncan, K., and Walsh, C. T. (1989). Nucleotide sequence of a cluster of
Escherichia coli enterobactin biosynthesis genes: identification of entA and
purification of its product 2,3 -dihydro-2,3 -dihydroxybenzoate dehydrogenase. Journal
of Bacteriology 171, 791-798.
Long, B. H., Carboni, J. M., Wasserman, A. J., Cornell, L. A., Casazza, A.-M., Jensen,
P. R., Lindel, T., Fenical, W., and Fairchild, C. R. (1998). Eleutherobin, a novel
cytotoxic agent that induces tubulin polymerization is similar to paclitaxel (Taxol).
Cancer Research 58, 1111-1115.
Luesch, H., Moore, R. E., Paul, V. J., Mooberry, S. L., and Corbett, T. H. (2001a).
Isolation of dolastatin 10 from the marine cyanobacterium Symploca species VP 642
and total stereochemistry and biological evaluation of its analogue symplostatin 1.
Journal of Natural Products 64, 907-910.
Luesch, H., Yoshida, W. Y., Moore, R. E., and Paul, V. J. (2000a). Isolation and
structure of the cytotoxin lyngbyabellin B and absolute configuration of lyngbyapeptin
A from the marine cyanobacterium Lyngbya majuscula. Journal of Natural Products
63, 1437-1439.
Luesch, H., Yoshida, W. Y., Moore, R. E., Paul, V. J., and Corbett, T. H. (2001b).
Total structure determination of apratoxin A, a potent novel cytotoxin from the marine
cyanobacterium Lyngbya majuscula. Journal of American Chemical Society 123,
5418-5423.
Luesch, H., Yoshida, W. Y., Moore, R. E., Paul, V. J., and Mooberry, S. L. (2000b).
Isolation, structure determination, and biological activity of Lyngbyabellin A from the
marine cyanobacterium Lyngbya majuscula. Journal of Natural Products 63, 611-615.
Maccabe, A. P., Vanliempt, H., Palissa, H., Unkles, S. E., Riach, M. B. R., Pfeifer, E.,
vonDohren, H., and Kinghorn, J. R. (1991). Delta-(L-Alpha-Aminoadipyl)-LCysteinyl-D-Valine Synthetase from Aspergillus-Nidulans - Molecular
Characterization of the Acva Gene Encoding the 1st Enzyme of the Penicillin
Biosynthetic Pathway. Journal of Biological Chemistry 266, 12646-12654.
MacNeil, D. J., Occi, J. L., Gewain, K. M., MacNeil, T., Gibbons, P. H., Ruby, C. L.,
and Danis, S. J. (1992). Complex organization of the Streptomyces avermitilis genes
encoding the avermeetin polyketide synthase. Gene 115, 119-125.
Manzanares, I., Cueves, C., Garcia-Nieto, R., Marco, E., and Gago, F. (2001).
Advances in the chemistry and pharmacology of ecteinascidins, a promising new class
of anti-cancer agents. Current Medicinal Chemistry and Anticancer Agents 1, 257276.
Marahiel, M. A., Stachelhaus, T., and Mootz, H. D. (1997). Modular peptide
synthetases involved in nonribosomal peptide synthesis. Chemical Reviews 97, 265 12673.
May, R. M. (2005). Biological diversity: differences between land and sea.
Philosophical Transactions of the Royal Society of London Series B-Biological
Sciences 343, 105-111.
McDaniel, R., Welch, M., and Hutchinson, C. R. (2005). Genetic approaches to
polyketide antibiotics. 1. Chemical Reviews 105, 543-558.
Milligan, K. E., Marquez, B. L., Williamson, R. T., and Gerwick, W. H. (2000).
Lyngbyabellin B, a toxic and antifungal secondary metabolite from the marine
cyanobacterium Lyngbya ma) uscula. Journal of Natural Products 63, 1440-3.
47
Miyazaki, K., Kobayashi, M., Natsume, T., Gondo, M., Mikami, T., Sakakibara, K.,
and Tsukagoshi, S. (1995). Synthesis and antitumor activity of novel dolastatin 10
analogs. Chemical & pharmaceutical bulletin 43, 1706-1718.
Moffitt, M. C. and Neilan, B. A. (2004). Characterization of the nodularin synthetase
gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins.
Applied and Environmental Microbiology 70, 6353-6362.
Molnar, I., Schupp, T., Ono, M., Zirkie, R., Milnamow, M., Nowak-Thompson, B.,
Engel, N., Toupet, C., Stratmann, A., Cyr, D. D., Gorlach, J., Mayo, J. M., Hu, A.,
Goff, S., Schmid, J., and Ligon, J. M. (2000). The biosynthetic gene cluster for the
microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So
ce9O. Chemistry & Biology 7, 97-109.
Moore, B. S., Hertweck, C., Hopke, J. N., Izumikawa, M., Kalaitzis, J. A., Nilsen, G.,
O'Hare, T., Piel, J., Shipley, P. R., Xiang, L., Austin, M. B., and Noel, J. P. (2002).
Plant-like Biosynthetic Pathways in Bacteria: From Benzoic Acid to Chalcone(1).
Journal of Natural Products 65, 1956-1962.
Moore, B. S. and Hopke, J. (2001). Discovery of new bacterial polyketide biosynthetic
pathway. Chembiochem 2, 35-38.
Moore,R.E. (1978). Algal nonisoprenoids. In Marine Natural Products, Chemical and
Biological Perspectives, P.3 .Scheuer, ed. (New York: Academic Press), pp. 43-124.
Moore,R.E. (1981). Constituents of the blue-green alga. In Marine Natural
Products:Chemical and Biological Perspectives, P.J.Scheuer, ed. (London: Academic
Press), pp. 1-52.
Moore, R. E. (1996). Cyclic peptides and depsipeptides from cyanobacteria: a review.
Journal of Industrial Microbiology 16, 134-143.
Motamedi, H., Cai, S. J., Shaflee, A., and Elliston, K. 0. (1997). Structural
organization of a multifunctional polyketide synthase involved in the biosynthesis of
the macrolide immunosuppressant FK506. European Journal of Biochemistry 244, 7480.
Mynderse, J. S., Moore, R. E., Kashiwagi, M., and Norton, T. R. (1977). Antileukemia
activity in the Oscillatoriaceae: isolation of debromoaplysiatoxin from Lyngbya.
Science 196, 53 8-540.
Nageli, C. (1849). Neue Denkschr. Allg. Schwiezer Ges. Naturwissenschaften 10, 1.
Nagle, D. G. and Paul, V. J. (1999). Production of secondary metabolites by
filamentous tropical marine cyanobacteria:ecological functions of the compounds.
Journal of Phycology 35, 1412-1421.
Nagle, D. G. and Paul, V. J. (1998). Chemical defense of a marine cyanobacterial
bloom. Journal of Experimental Marine Biology and Ecology 225, 29-3 8.
Newman, D. J. and Cragg, G. M. (2004b). Marine Natural Products and Related
Compounds in Clinical and Advanced Preclinical Trials. Journal of Natural Products
67, 1216-1238.
Newman, D. J. and Cragg, G. M. (2004a). Advanced preclinical and clinical trials of
natural products and related compounds from marine sources. Current Medicinal
Chemistry 11, 1693-1713.
Newman, D. J., Cragg, G. M., and Snader, K. M. (2003). Natural products as sources
of new drugs over the period 1981-2002. Journal of Natural Products 66, 1022-1037.
Newman, D. J., Cragg, G. M., and Snader, K. M. (2000). The influence of natural
products upon drug discovery. Natural Product Reports 17, 215-234.
Orjala, J., Nagle, D. G., Hsu, V., and Gerwick, W. H. (1995). Antillatoxin, an
exceptionally ichthyotoxic cyclic lipopeptide from the tropical cyanobacterium
Lyngbya majuscula. Journal of American Chemical Society 117, 828 1-8282.
Osborne, N. J., Webb, P. M., and Shaw, G. R. (2001). The toxins of Lyngbya
majuscula and their human and ecological health effects. Environment International
27, 38 1-392.
Paerl,H.W. (1988). Growth and reproduction strategies of freshwater blue-green algae
(cyanobacteria). In Growth and reproductive strategies of freshwater phytoplankton,
C.D.Sandgren, ed. (Cambridge: Cambridge University Press), pp. 261-3 15.
Perez-Baz, J., Canedo, L. M., Fernandez Puentes, J. L., and Silva Elipe, M. V. (1997).
Thiocoraline, a novel depsipeptide with antitumor activity produced by a marine
Micromonospora. II. Physico-chemical properties and structure determination. Journal
of Antibiotics (Tokyo) 50, 738-741.
Pettit, G. R., Herald, C. L., Doubek, D. L., Delbert, A. L., Arnold, E., and Clardy, J.
(1982). Isolation and structure of bryostatin 1. Journal of American Chemical Society
104, 6846-6848.
Pettit, G. R., Kamano, Y., Herald, C. L., Tuinman, A. A., Boettner, F. E., Kizu, H.,
Tomer, K. B., and Bontems, R. J. (1987). The Isolation and Structure of a Remarkable
Marine Animal Antineoplastic Constituent: Dolastatin 10. Journal of American
Chemical Society 109, 6883-6885.
Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E., and Khosla, C. (2001).
Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli.
Science 291, 1790-1792.
Pfeifer, B. A. and Khosla, C. (2001). Biosynthesis of polyketides in heterologous
hosts. Microbiology and Molecular Biology Reviews 65, 106-118.
Pfeifer, B. A., Wang, C. C., Walsh, C. T., and Khosla, C. (2003). Biosynthesis of
Yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as
a heterologous host. Applied and Environmental Microbiology 69, 6698-6702.
Piel, J. (2004). Metabolites from symbiotic bacteria. Nat Prod Rep 21, 519-538.
Piel, J., Hertweck, C., Shipley, P. R., Hunt, D. M., Newman, M. S., and Moore, B. S.
(2000). Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster
from the marine isolate 'Streptomyces maritimus': evidence for the derailment of an
aromatic polyketide synthase. Chemistry & Biology 7, 943-955.
Proteau, P. J., Gerwick, W. H., Garcia-Pichel, F., and Castenholz, R. (1993). The
structure of scytonemin, an ultraviolet sunscreen pigment from the sheaths of
cyanobacteria. Experientia 49, 825-829.
Rinehart, K. L., Holt, T. G., Fregeau, N., Stroh, J. G., Keifer, P. A., Sun, F., Li, H. L.,
and Martin, D. G. (1990). Ecteinascidins 729, 743, 745, 759A, 759B, and 770: potent
antitumor agents from the Caribbean tunicate Ecteinascidia turbinata. Journal of
Organic Chemistry 55, 4512-4515.
Rinehart, K. L. and Lithgow-Bertelloni, A. M. Dehydrodidemnin B. WO 1990GB1495{WO 9104985], 1-41. 1991.
Ref Type: Patent
Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., and Stainier, R. Y. (1971).
Generic assignments, strain histories and properties of pure cultures of cyanobacteria.
Journal of General Microbiology 111, 1-61.
Romero, F., Espliego, F., Perez-Baz, J., de Quesada, G., Gravalos, D., De la Calle, F.,
and Fernandez Puentes, J. L. (1997). Thiocoraline, a new depsipeptide with antitumor
activity produced by a marine Micromonospora. I. Taxonomy, fermentation, isolation,
and biological activities. Journal of Antibiotics (Tokyo) 50, 734-73 7.
Rouhiainen, L., Paulin, L., Suomalainen, S., Hyytiainen, H., Buikema, W., Haselkorn,
R., and Sivonen, K. (2000). Genes encoding synthetases of cyclic depsipeptides,
anabaenopeptilides, in Anabaena strain 90. Molecular Microbiology 37, 156-67.
Salomon, C. E., Magarvey, N. A., and Sherman, D. H. (2004). Merging the potential
of microbial genetics with biological and chemical diversity: an even brighter future
for marine natural product drug discovery. Natural Product Reports 21, 105-12 1.
50
Schopf,J.W. (2005). The fossil record: Tracing the roots of cyanobacterial lineage. In
The ecology of cyanobacteria: Their diversity in time and space, B.A.Whitton and
M.Potts, eds. (Netherlands: Kiuwer Academic Publishers), pp. 13-35.
Schwarzer, D., Finking, R., and Marahiel, M. A. (2003). Nonribosomal peptides: from
genes to products. Natural Product Reports 20, 275-287.
Schwecke, T., Aparicio, J. F., Molnar, I., Konig, A., Khaw, L. E., Haydock, S. F.,
Oliynyk, M., Caffrey, P., Cortes, J., Lester, J. B., and et al. (1995). The biosynthetic
gene cluster for the polyketide immunosuppressant rapamycin. Proceedings of the
National Academy of Sciences of the United States of America 92, 7839-7843.
Seo, P.-L. and Yokota, A. (2003). The phylogenetic relationships of cyanobacteria
inferred from 16S rRNA, gyrB, rpoCl and rpoDl gene sequences. Journal of General
and Applied Microbiology 49, 191-203.
Sieber, S., Linne, U., Hillson, N., Roche, E., Walsh, C., and Marahiel, M. (2002).
Evidence for a monomeric structure of nonribosomal Peptide synthetases. Chemistry
& Biology 9, 997.
Sieber, S. A. and Marahiel, M. A. (2005). Molecular mechanisms underlying
nonribosomal peptide synthesis: approaches to new antibiotics. Chemical Reviews
105, 715-738.
Sieber, S. A. and Marahiel, M. A. (2003). Learning from Nature's drug factories:
Nonribosomal peptide synthesis of macrocyclic peptides. Journal of Bacteriology 185,
7036-7043.
Sieber, S. A., Walsh, C. T., and Marahiel, M. A. (2003). Loading peptidyl-coenzyme
A onto peptidyl carrier proteins: A novel approach in characterizing macrocyclization
by thioesterase domains. Journal of American Chemical Society 125, 10862-10866.
Silakowski, B., Kunze, B., Nordsiek, G., Blocker, H., Hofie, G., and Muller, R.
(2000). The myxochelin iron transport regulon of the myxobacterium Stigmatella
aurantiaca Sg a15. European Journal of Biochemistry 267, 6476-6485.
Silakowski, B., Schairer, H. U., Ebret, H., Kunze, B., Weinig, S., Nordsiek, G.,
Brandt, P., Blocker, H., Hofle, G., Beyer, S., and Muller, R. (1999). New lessons for
combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene
cluster of Stigmatella aurantiaca DW4/3-1. Journal of Biological Chemistry 274,
37391-37399.
Simmons, T. L., Adrianasolo, E., McPhail, K., Flatt, P., and Gerwick, W. H. (2005).
Marine natural products as anticancer drugs. Mol Cancer Ther 4, 333-342.
51
Sings, H. L. and Rinehart, K. L. (1996). Compounds produced from potential tunicateblue-green algal symbiosis: a review. Journal of Industrial Microbiology 17, 385-396.
Solomon, A. E. and Stoughton, R. B. (1978). Dermatitis from purified sea algae toxin
(debromoaplysiatoxin). Archives of Dermatology 114, 1333-5.
Sone, H., Kondo, T., Kiruyu, M., Ishiwata, H., Ojika, M., and Yamada, K. (1995).
Dolabellin, a Cytotoxic Bisthiazole Metabolite from the Sea Hare Dolabella
auricularia: Structural Determination and Synthesis. Journal of Organic Chemistry 60,
4774-4781.
Stachelhaus, T., Mootz, H. D., and Marahiel, M. A. (1999). The specificity-conferring
code of adenylation domains in nonribosomal peptide synthetases. Chemistry &
Biology 6, 493-505.
Stanier, R. Y., Sistrom, W. R., Hansen, T. A., Whitton, B. A., Castenholz, R. W.,
Pfenning, N., Gorlenko, V. N., Kondratieva, E. N., Eimhjellen, K. B., Whittenbury, R.,
Gherna, R. L., and Truper, H. G. (1978). Proposal to place the nomenclature of the
cyanobacteria (blue-green algae) under the rules of the International Code of
Nomenclature of Bacteria. International Journal of Systematic Bacteriology 28, 335336.
Staunton, J., Caffrey, P., Aparicio, J. F., Roberts, G. A., Bethell, S. S., and Leadlay, P.
F. (1996). Evidence for a double-helical structure for modular polyketide synthases.
Nature Structural Biology 3, 188-92.
Staunton, J. and Weissman, K. J. (2001). Polyketide biosynthesis: a millennium
review. Natural Product Reports 18, 380-416.
Stevenson, C. S., Capper, E. A., Roshak, A. K., Marquez, B., Eichman, C., Jackson, J.
R., Mattern, M., Gerwick, W. H., Jacobs, R. S., and Marshall, L. A. (2002a). The
identification and characterization of the marine natural product scytonemin as a novel
antiproliferative pharmacophore. Journal of Pharmacology and Experimental
Therapeutics 303, 858-866.
Stevenson, C. S., Capper, E. A., Roshak, A. K., Marquez, B., Grace, K., Gerwick, W.
H., Jacobs, R. S., and Marshall, L. A. (2002b). Scytonemin--a marine natural product
inhibitor of kinases key in hyperproliferative inflammatory diseases. Inflammation
Research 51, 112-114.
Takashima, M. and Sakai, H. (1960). A new toxic substance, teleocidin, produced by
Streptomyces. II. Biological studies of teleocidin. Bulletin of the Agricultural
Chemical Society of Japan 24, 652-655.
52
Taraboletti, G., Poli, M., Dossi, R., Manenti, L., Borsotti, P., Faircioth, G., Broggini,
M., D'Inalci, M., Ribatti, D., and Goavazzi, R. (2004). Antiangiogenic activity of
aplidine, a new agent of marine origin. Bristish Journal of Cancer 90, 2418-2424.
Thacker, R. W. and Paul, V. J. (2004). Morphological, chemical and genetic diversity
of the tropical cyanobacteria Lyngbya spp. and Symploca spp. (Oscillatoriales).
Applied and Environmental Microbiology 70, 3305-3312.
Tillett, D., Dittmann, E., Erhard, M., von Dohren, H., Borner, T., and Neilan, B. A.
(2000). Structural organization of microcystin biosynthesis in Microcystis aeruginosa
PCC7806: an integrated peptide-polyketide synthetase system. Chemistry & Biology
7, 753-764.
Towle, M. J., Salvato, K. A., Budrow, J., Wels, B. F., Kuznetsov, G., Aalfs, K. K.,
Welsh, S., Zheng, W., Seletsk, B. M., Palme, M. H., Habgood, G. J., Singer, L. A.,
DiPietro, L. V., Wang, Y., Chen, J. J., Quincy, D. A., Davis, A., Yoshimatsu, K.,
Kishi, Y., Yu, M. J., and Littlefield, B. A. (2001). In vitro and in vivo anticancer
activities of synthetic macrocyclic ketone analogues of halichondrin B. Cancer
Research 61, 1013-1021.
Trauger, J. W., Kohli, R. M., and Walsh, C. T. (2001). Cyclization of backbonesubstituted peptides catalyzed by the thioesterase domain from the tyrocidine
nonribosomal peptide synthetase. Biochemistry 40, 7092-8.
Venter, J. C., Remington, K., Heidelberg, J. F., Halpern, A. L., Rusch, D., Eisen, J. A.,
Wu, D., Paulsen, I., Nelson, K. E., Nelson, W., Fouts, D. E., Levy, S., Knap, A. H.,
Lomas, M. W., Nealson, K., White, 0., Peterson, J., Hoffman, J., Parsons, R., BadenTillson, H., Pfannkoch, C., Rogers, Y. H., and Smith, H. 0. (2004). Environmental
genome shotgun sequencing of the Sargasso Sea. Science 304, 66-74.
Walsh, C. T. (2004). Polyketide and nonribosomal peptide antibiotics: modularity and
versatility. Science 303, 1805-18 10.
Watanabe, K., Wang, C. C., Boddy, C. N., Cane, D. E., and Khosla, C. (2003).
Understanding substrate specificity of polyketide synthase modules by generating
hybrid multimodular synthases. Journal of Biological Chemistry 278, 42020-42026.
Weber, G., Schorgendorfer, K., Schneiderscherzer, E., and Leitner, E. (1994). The
Peptide Synthetase Catalyzing Cyclosporine Production in Tolypocladium Niveum Is
Encoded by a Giant 45.8-Kilobase Open Reading Frame. Current Genetics 26, 120125.
Weissman, K. J. (2004). Polyketide biosynthesis: understanding and exploiting
modularity. Philosophical Transactions of the Royal Society of London Series AMath, Physics and Engineering Sciences 362, 2671-2690.
53
Whitton,B.A. and Potts,M. (2000). Introduction to cyanobacteria. In The ecology of
cyanobacteria. Their diversity in time and space., B.A.Whitton and M.Potts, eds.
(Dordrecht: Kluwer Academic Publisher), pp. 1-11.
Wilmotte,A. (1994). Molecular evolution and taxanomy of cyanobacteria. In The
Molecular Biology of Cyanobacteria, D.A.Bryant, ed. (Netherlands: Kluwer Academic
Publishers), pp. 1-25.
Wipf, P., Reeves, J. T., and Day, B. W. (2004). Chemistry and biology of curacin A
2. Current Pharmaceutical Design 10, 1417-1437.
Wu, M. Novel bioactive secondary metabolites from the marine cyanobacterium
Lyngbya majuscula. 1996. Oregon State University.
Ref Type: Thesis/Dissertation
Wu, M., Okino, T., Nogle, L. M., Marquez, B. L., Williamson, R. T., Sitachitta, N.,
Berman, F. W., Murray, T. F., McGough, K., Jacobs, R., Colsen, K., Asano, T.,
Yokokawa, F., Shioiri, T., and Gerwick, W. H. (2000). Structure, Synthesis, and
Biological Properties of Kalkitoxin, a Novel Neurotoxin from the Marine
Cyanobacteriurn Lyngbya majuscula. Journal of American Chemical Society 122,
12041-12042.
Wu, N., Tsuji, S. Y., Cane, D. E., and Khosla, C. (2001). Assessing the Balance
between Protein-Protein Interactions and Enzyme- Substrate Interactions in the
Channeling of Intermediates between Polyketide Synthase Modules. Journal of
American Chemical Society 123, 6465-6474.
54
CHAPTER II
ISOLATION OF A CRYPTIC GENE CLUSTER FROM THE
CYANOBACTERIUM LYNGBYA MAJUSCULA JHB AND
DEMONSTRATION OF ITS EXPRESSION
INTRODUCTION
Marine cyanobacteria have proven to be one of the most versatile producers of
secondary metabolites and many of these metabolites demonstrate antiproliferative,
cytotoxic or neurotoxic activity, making them invaluable as potential therapeutic leads
or pharmacological tools. Hectochiorin (54) and jamaicamides A-C (27-29) were
isolated as major components of a Jamaican collection of the cyanobacterium,
Lyngbya majuscula (designated as L. majuscula strain JHB) (Edwards et al., 2004;
Marquez et al., 2002).
Hectochlorin (54) possesses structurally intriguing features such as the
presence of gem-dichioro group and two dihydroxyisovaleric acid units. It was also
found to be possess potent antifungal and cytotoxic properties (Marquez et al., 2002).
Ptk2 cells (derived from the rat kangaroo, Potorous tridactylus) treated with
hectochiorin showed an increase in the number of binucleated cells as a result of the
arrest of cytokinesis, a finding typical of compounds that cause hyperpolymerization
of actin. The unique biological properties and presence of intriguing structural
features in hectochiorin, such as the gem-dichloro group and two 2,3dihydroxyisovaleric acid units, prompted us to attempt the cloning and
characterization its biosynthetic gene cluster to obtain details regarding its
biosynthesis at the molecular genetic level.
55
O
O
0
27
0
21)j__8
-
24
HO
16..J
1"O
S2O
25
Hectochlorin (54)
HfNJLL
OCH3
o
N
R=BrJaniaicamide A(27)
R=H=3amaicamide B (28)
HNAyk
OCH3
N
O
Jamaicade C (29)
The structure of hectochiorin (54) suggested that it was derived from a
PKS/NRPS pathway, a characteristic pathway for metabolites derived from L.
majuscula. In order to isolate the hectochiorin biosynthetic gene cluster, we decided
to employ a general PCR method (Turgay and Marahiel, 1994) and amplify NRPS
genes from L. majuscula JHB genomic DNA using degenerate primers based on core
motifs (Neilan et al., 1999). However, this approach led to the identification of an
approximately 20 kb NRPS/PKS gene cluster whose genetic architecture suggested its
involvement in the biosynthesis of a metabolite unrelated to hectochiorin (54).
Because the metabolite for which this cluster codes the biosynthesis remains as yet
uncharacterized, it was designated as a "cryptic cluster" (Bentley et al., 2002; Omura
et al., 2001; Zazopoulos et al., 2003).
Herein, the cloning and sequence characterization of the cryptic gene cluster
along with demonstration of its expression from the cultured L. majuscula JHB is
described. A novel interdisciplinary approach, designed to identify the metabolite
produced by this cryptic cluster, is also discussed. Genomics guided approaches, such
as the one discussed here, will become increasingly important in natural products
research as a complement to the traditional methods for the isolation of novel natural
products.
57
RESULTS AND DISCUSSION
Isolation of genonuc DNA from Lyngbya majuscula JHB
The first step towards generating a genomic library of Lyngbya majuscula JHB
is the isolation and purification of high molecular weight genomic DNA. We were
also interested in surveying several DNA isolation procedures in order to optimize a
simplified protocol for the isolation of high molecular weight DNA from samples
immediately after their collection in the field. In addition to L. majuscula JHB, three
other strains of L. majuscula (L. majuscula l9L, Lyngbya NPI 03 and Lyngbya MNT
01) and a Phormidium sp. were used in the various methods that were explored. L.
majuscula JHB, 19L and Phormidium were maintained as unialgal cultures in our
laboratory whereas Lyngbya NPI and Lyngbya MNT were field collected samples
stored at -20°C in a mixture of isopropanol and water for future chemical extraction
purposes (Experimental).
Several methods were explored in order to optimize a protocol that consistently
yielded intact high molecular weight DNA in large concentrations and in a form
suitable for subsequent manipulations and analysis. The first method used for the
isolation of genomic DNA was designed for filamentous fungi (M. Cone, personal
communication). This method resulted in low recovery of genomic DNA from L.
majuscula JHB, L. majuscula 19L and Phormidium sp., and genomic DNA obtained
from Lyngbya MNT 01 and NPI 03 appeared to be completely degraded (Figure
II.1A). DNA obtained by a second method using CTAB-buffer resulted in isolation of
low molecular weight genomic DNA. Moreover, this method could not be scaled-up
for higher yields of genomic DNA. Finally for library construction, genomic DNA
from L. majuscula JHB was isolated using a previously described protocol (Sambrook
et al., 1989) (Figure II.1B). However, a major drawback with this method was
carryover contamination of phenol in DNA samples. The presence of phenol was
undesirable because it is known to interfere with downstream enzymatic manipulation
of DNA. Therefore, this method was modified further to include a purification step
after phenol extraction using Qiagen's 100IG Genomic Tip DNA Kit. The only
disadvantage with this method was low recovery of DNA and therefore the need for
large amounts of DNA at the start of the procedure (- 5 pjg). Nonetheless, DNA
obtained after this purification step was high molecular weight and free of phenol.
XHindlll 2
AMarker
12
34
B
5
40kb
C
Genomic DNA
L. majuscula JHB
XHindlll 2
Marker
Genomic DNA 1740kb
(L.
majuscula JHB) control
-
kHindIII 2
Marker
22kb
Figure 11.1: Isolation of high molecular weight genomic DNA. A. High molecular
weight isolated by using CTAB-buffer from Lyngyba MNTO1 (1), Lyngbya NPIO3
(2), L. majusucla JHB (3) Phormidium (4) and L. majuscula 1 9L (5). B. High
molecular weight genomic DNA was isolated from L. majuscula JHB by
extraction with phenol followed by further purification using Qiagen's G/100
Genome Tip Kit. C. L. majuscula JHB genomic DNA isolated using DNeasy Plant
Kit.
DNA isolation using several commercially available kits namely, DNeasy
Plant Mini Kit (Qiagen), DNAzol (Gibco Life Sciences) and Wizard DNA Purification
Kit (Promega) was also investigated. DNA isolation using DNeasy Plant kit had some
distinct advantages because the DNeasy shredder column supplied with the kit was
59
designed to remove cellular debris, particularly polysaccharides from a viscous lysate,
making recovery of genomic DNA more efficient. Although this method resulted in
the isolation of good quality genomic DNA, it was not of high molecular weight (< 22
kb) and was therefore unsuitable for library construction; however, it was very
efficient for use as template for PCR (Figure II.1C). The Wizard kit was also fairly
effective but the DNAzo1 reagent proved to be inefficient in recovering genomic DNA
from all samples used in the study.
Isolation of genomic DNA from the various cyanobacterial strains proved to be
challenging. The presence of extracellular mucilage, pigments and filamentous
morphology of some cyanobacteria are among the reasons for poor recovery of DNA
from these organisms (Tillett and Neilan, 2000). The method adapted from Sambrook
et al. (1986) (Method III, Experimental) proved to be most efficient for the isolation of
intact high molecular weight DNA. Lysis with liquid nitrogen along with prolonged
digestion with proteinase K at 50°C probably facilitated efficient lysis for the recovery
of high molecular weight intact genomic DNA which was ultimately used for library
construction. There were slight variations with regard to yield of genomic DNA from
the various strains and higher concentrations were obtained from L. majuscula JHB
and L. majuscula 1 9L when compared to the Phormidium sp. The presence of copious
amounts of mucilaginous sheath material and bioflim-flim like colony morphology of
some Phormidium sp. are believed to hinder cell lysis and purification of nucleic acids
(Fiore et al., 2000).
Genomic DNA obtained from the two preserved strains was completely
degraded and this suggested that the mode of tissue preservation has a marked effect
on the quality of nucleic acids. A simple method without the use of liquid nitrogen or
organic solvents could not be established for isolation of genomic DNA during field
collections. However, we discovered that storage of samples in RNA Later TM
(Ambion) for transport to the laboratory preserved the quality of DNA without
affecting yield thus obviating the need for such a protocol.
PCR amplification of probes
The structure of hectochlorin suggested that it was derived from a hybrid
PKS/NRPS pathway. While Cl -C8 were hypothesized to be derived from acetate
units assembled by PKS's, the other two units were proposed to be derived from the
amino acid cysteine and 2,3-dihydrdxyisovaleric acid (DHiv), a precursor in valine
biosynthesis (Figure 11.2). In order to isolate the hectochiorin biosynthetic gene
cluster, we decided to employ a general PCR method (Turgay and Marahiel, 1994) and
amplify NRPS/PKS genes from L. majuscula JHB genomic DNA using degenerate
primers based on core motifs. Our goal was then to select adenylation domains that
were specific for valine and cysteine and use them as probes to screen the library.
CI
II
0
20
Acetate
OH
15
Figure 11.2: Proposed biosynthetic precursors of hectochiorin. Precursor feeding
studies have demonstrated that the C1-C8 polyketide and C26-C27 derive from
acetate, while the pendant methyl C9 is derived from SAM. Biosynthetic feeding
studies to investigate the origins of the two DHiv units were inconclusive (Nogle,
2002).
61
The degenerate primers used for this purpose were designed based on
conserved peptide synthetase motifs from several bacteria and fungi. Additionally,
they were designed to favor codon bias of cyanobacterial genes (Neilan et al., 1999).
The primers A2-Forward based on the adenylation domain AGGAVYP core motif I
and A8-Reverse based on the QVKIRG adenylation domain core motif V
(Experimental) were used to amplify an approximately 1.2 kb fragment from L.
majuscula
genomic DNA (Figure 11.3). This PCR product was purified and cloned
into pGEM-T Easy vector and clones were screened by PCR using SP6 and T7-P
primers. PCR products were digested with the frequent cutting enzyme TaqI, and
based on their restriction digest pattern; clones were grouped into four distinct groups,
I-IV. BLAST analysis of representatives of each group revealed that they were all
peptide synthetases with highest homology to nostopeptolide NosD (54% identity)
(Hoffmann et al., 2003) and anabaenopeptilide ApdA (53% identity) (Rouhiainen et
al., 2000). In order to determine the substrate specificity of the various clones, a
bioinformatics-based approach developed by Stacheihaus et al. and Challis et al. was
used in which clones from each group were translated and key amino acids involved in
substrate recognition and binding were identified by alignment with GrsA-Phe
adenylation domain (Stachelhaus et al., 1999; Challis et al., 2000). Based on this
analysis, group I clones were predicted to activate valine, clones from group II had a
predicted specificity for proline, group III for asparagine and IV for leucine (Table
11.1). A single clone (Val-Aden-20) from Group 1 was selected as a probe and was
labeled with dioxigenin (DIG) for library screening.
62
Figure 11.3: Amplification of NRPS A-domain probe. Degenerate primers
based on A2 and A8 adenylation domain core motifs were used to amplify an
approximately 1.2 kb fragment from L. majuscula JHB genomic DNA.
Table 11.1: Substrate specificity of NRPS A-domain probes. The amino acid
residues involved in substrate binding and recognition were determined using the
method of Challis et al. (2000) for representatives from each group of unique clones
digested with TaqI.
Group
Position of binding pocket amino acids
235 236 239 278 299 301 322 330
Predicted amino acid
substrate
I
D
A
L
W
L
G
G
T
valine
II
D
F
Q
F
A
H
V
proline
III
D
L
T
K
I
I
G
H
V
asparagine
IV
D
A
W
F
L
G
N
V
leucine
63
In order to focus on an adenylation domain probe with predicted specificity for
cysteine, a pair of primers was designed based on alignments of several previously
characterized cysteine adenylation domains. Thus, HMWP from Yersinia pestis
(Gehring et al., 1998), bacitracin BacA from Bacillus lichenformis (Konz et al., 1997)
epothilone EpoB from Sorangium cellulosum (Molnar et al., 2000), and pyochelin
PchE and PchF from Pseudomonas aeruginosa (Quadn et al., 1999) were aligned to
design primers 720F and 840R. (Chang et al., 2002) (Figure 11.4).
HMWP2
BacA
EpoB
PchE
PchF
752
708
708
694
731
DRVLALSALHFDLSVYDI
DRVLALSSLSFDLSVYDV
DRVLALSSLSFDLSVYDV
DRVLGLAELSFDLSVYDF
DRLLAVSALDFDLSVFDT
720F
---------------------------------------------------------------------------------
HWRSIPYGFPLTNQRY 896
SWASIPYGNPLRNQTF 852
SWASIPYGRPLRNQTF 852
ELASIPYGRALRGQSV 839
HWRSIPYGRALPGQAY 874
4840R
Figure 11.4: ClustaiW alignment of cysteine adenylation domains. High molecular
weight 2 (HMWP), bacitracin BacA, epothiolone (EpoP), pyochelin PchE and PchF
(Quadri et al., 1999). Conserved amino acids for cysteine adenylation were used to
design primers 720F and 840R.
PCR amplification using this primer pair 720F and 840R resulted in the
amplification of an approximately 400 bp fragment which was purified and cloned
into pGEM-T Easy vector for sequencing (Figure 11.5). Several clones were
sequenced and these results revealed that all the clones were identical which led to the
conclusion that this primer pair favored the amplification of a single adenylation
domain due to PCR bias. BLAST analysis revealed that it was most similar to EpoB
(57% identity) (Molnar et al., 2000). The substrate specificity determined by aligning
translated sequences with GrsA-Phe adenylation domain also revealed that it was
predicted to activate cysteine (Table II. 2) (Challis et al., 2000). Based on these
results, a single clone Cys-Aden-9 was purified and labeled with DIG for library
screening.
64
720F/840R
Marker
product
Figure 11.5: Amplification of cyteine-specific A-domain. PCR amplification
using the degenerate primers, 720F and 840R resulted in the amplification of an
approximately 400 bp product from L. majuscula JHB genomic DNA.
Table 11.2: Substrate specificity of cysteine A-domain probe. The amino acid
residues involved in substrate binding and recognition were determined using
the method of Challis et al. (2000) for the adenylation domain fragment
amplified using 720F/840R.
Group
Cys-Aden
Position of binding pocket amino acids
235 236 239 278 299 301 322 330
L
D
Y
I G I V
N
Predicted
amino acid
substrate
cysteine
Polyketide synthase probes spanning a small region of the ketosynthase
domain (KS) were amplified using previously described primers (Beyer et al., 1999).
The approximately 700 bp product was cloned into pGEM-T Easy and sequenced. All
sequences showed high homology to known polyketide synthases such as epothilone
EpoF (58% identity) (Molnar et al., 2000) myxothiazol MtaE (55%) (Silakowski et al.,
1999) and microcystin McyD (53% identity) (Tillett et al., 2000). A "mixed" pool of
KS fragments was used for probe generation and was obtained by purification of the
PCR product from genomic DNA. The PCR product was purified and labeled with
DIG.
Figure 11.6: Amplification of KS domain probe. PCR amplification using the
degenerate primers, KSUp and KSD1 resulted in the amplification of a 700 bp
fragment of the 3-ketosynthase domain from L. majuscula JHB genomic DNA.
Construction and screening of a genomic DNA library
Once a method for the isolation of good quality genomic DNA was
established, our next goal was the construction of a library that offered full coverage
of the L. majuscula genome whose size was maximally estimated to be around 10 Mb
based on the genome sizes ofNostocpunctiforme (Meeks et al., 2001) and Anabaena
PCC712O (Kaneko et al., 2001). Therefore, with an average insert size of about 40 kb
we aimed to screen at least 2500 clones in order to obtain adequate coverage of the
genome (approximately 8-fold). Several vectors for library construction were
considered and our initial choice was the cosmid vector pOJ446 (Bierman et al., 1992)
because it has been used for the isolation of the barbamide and curacin A biosynthetic
gene clusters (Chang et al., 2002; Chang et al., 2004). However, this vector was found
to be unstable in E. coli and could not be purified from glycerol stocks (P. Flatt,
personal communication). Therefore, we decided to use the pWEB::TNC cosmid
cloning kit from Epicentre Technology which was designed to hold inserts of
approximately 40 kb.
Next, isolated and purified genomic DNA was subjected to size selection in a
0.7 % agarose gel. However, recovering sufficient DNA from the gel after size
selection proved to be a challenge, and several concentrations of the Gelase® enzyme
and buffer had to be tested in order to maximize recovery of end-repaired genomic
DNA from the gel. We found that equilibration of the gel slice in excess Gelase buffer
(5 volumes) prior to overnight digestion with the Gelase® enzyme resulted in
increased recovery of DNA. The genomic DNA was then ligated into cosmid
pWEB::TNC for construction of the library. The titer of the library generated was
approximately 2.2 x
iO3
which was screened with the three DIG-labeled probes were
discussed previously by colony hybridization techniques. This resulted in the
identification of 53 cosmid clones which probed positive with the KS-domain probe
and 30 clones which probed positive with both of the NRPS probes. Of these 83
clones, only eleven probed positive with both PKS and NRPS probes and these were
selected for further analysis by restriction mapping and Southern hybridization.
Southern analysis of the eleven cosmids led to the identification of two, designated as
67
1-105 and 111-105, which hybridized strongly to all three probes. Restriction digest of
the two cosmids with EcoRI, HindIlI, BamHI, NcoI and PstI revealed that they had
similar restriction patterns and were likely overlapping DNA fragments. This led us to
speculate that these two cosmids might harbor different regions of the putative
hectochiorin biosynthetic gene cluster and thus they were selected for shot-gun
sequencing. The total size of each cosmid was estimated to be approximately 41 kb
(1-105) and 49 kb (111-105).
EcoRI
HindIil BarnHI NcoI
PsS
DIG-lobeled
A lAndiS
AB
AB
AB
ABAB
2,narker
B
EcoR
KS positive DIG-lobeled
control
yorker
AB
b
21 kb
HirtdIII BamHI NcoI
Psti
AB
AB
AB
AB
Cys-eden
EcoRl HindIIIBamHI
NcoI
PsLI
DIG-lebeled
merirer A B
AB
AB
AB
AB
21 kb
5 I kb
5 1 kL
35 b
35kb
-
2 0kb
*0
20k'
Figure 11.7: Restriction digest analyses (A) and Southern hybridizations of cosmids
1-105 and 111-105 (B). A. Restriction digest of cosmids 1-105 (A) and 111-105 (B)
show that they are overlapping fragments. B. Southern analysis using ketosynthase
domain probe (left) and cysteine adenylation domain probe confirmed that they were
overlapping cosmids (right).
LSI
In order to verify the accuracy of Southern analyses, a PCR screen of the two
cosmids employing primers, KS-Up/KS-Dl and 720F/840R, was performed. The
resulting amplicons were cloned into pGEM-T vector and sequenced. Most of the
clones obtained with the 720F/840R primers were most similar to EpoB (57%
identity) (Molnar et al., 2000) and confirmed the presence of a domain likely involved
in the incorporation of cysteine in both cosmids. Results with the ketosynthase
domain primer products were mixed in that a large fraction of clones sequenced were
similar to a putative L-glutamine: D-fructose aminotranferase from from Shigella
flexneri
(70%) (NP_709542) and only one clone was similar to MtaD (46% identity)
(Silakowski et al., 1999).
Based on this data, cosmids 1-105 and 111-105 were chosen as candidates for
sequencing. However, during subsequent purification of the two cosmids for shotgun
cloning, we observed that cosmid 111-105 had undergone a rearrangement and was
missing several EcoRI fragments that were initially observed. There are several
reasons for cosmid instability including genotype of the host strain, the presence of
short tandem repeats (Song et al., 2001) and copy number of the vector. It has been
observed that cosmid stability is improved in recA mutant host strains (Silby and
Mahanty, 2000). Because theE. coli strain EPI100 used in this study is a recA mutant,
the high copy number of the cosmid is likely the cause for the observed instability.
Shot-gun cloning and sequencing of cosmid 1-105
For sequencing of cosmid 1-105, two main sub-cloning approaches were
explored. In the first method, cosmid DNA was sheared by sonication and fragments
ranging from 0.75 kb-i .5 kb were size selected, end-repaired and ligated into the
prepared pGEM3Zf (+) vector. The second approach consisted of digesting the
cosmid partially with a frequent cutting enzyme, like AluI and Sau3Al, followed by
selection of fragments in the right size range and ligation into pGEM3Zf (+) or
pBluescript, respectively. Upon sequencing the sub-clones, we observed that a large
fraction of clones obtained by the first method had insert sizes < 750 bp which was not
conducive for the assembly of large contiguous stretches of DNA. However,
subclones obtained by the latter method contained fragments that were larger and
more suitable for assembly.
Sequence analysis of gene cluster
The DNA sequence of cosmid 1-105 was determined by shotgun cloning and
assembled into a roughly 20 kb contiguous stretch of DNA. The overall G+C content
in this region is approximately 45% which is consistent with the G+C content
encountered in cyanobacterial genomes (www.kazusa.or.ip/cyano) and other
secondary metabolite gene clusters from L. majuscula (Chang et al., 2002; Chang et
al., 2004; Edwards and Gerwick, 2004; Edwards et al., 2004). Analysis of the 20 kb
region revealed that it consists of three ORF '5. Preliminary analysis of the largest
ORF revealed that it was a mixed NRPS/PKS gene cluster and that it contained several
features that suggested its involvement in the biosynthesis of a metabolite unrelated to
hectochlorin (54) (Figure II.8B). Previous chemical investigations revealed the
presence of only four major compounds in this strain of Lyngbya majuscula (Edwards
et aL, 2004; Marquez et al., 2002) and none of these is consistent with the gene cluster
present on 1-105. Therefore, this cluster was designated as "cryptic gene cluster",
which has been defined as a gene cluster for which a corresponding metabolite
remains as yet unidentified. A survey of all literature reporting the presence or
isolation of cryptic gene clusters from several organisms revealed that a uniform
system of nomenclature of these clusters does not exist (Bentley et al., 2002; Challis
and Ravel, 2000; Omura et al., 2001; Zazopoulos et al., 2003; McAlpine et al., 2005).
Therefore, we propose here a system that will identify these clusters with first letter of
genus and first letter of species followed by cry (cryptic gene cluster). Hereafter,
according to the proposed nomenclature, this cryptic gene cluster will be referred to as
LMcryl (ngbyarnajuscula yptic gene cluster).
70
A
ORF
LMcryl
II
III
>c
20kb
B
LMcryl
NRPS
Module I
NRPS
Module 2
NRPS
Module 3
F;S
Module 4
Figure 11.8: Genetic architecture of LMcryl and modular organization of
LMcryl. A 20 kb contiguous region sequenced from cosmid 1-105 was
shown to contain three open reading frames, LMciyl , ORF II and ORF
III. (A). Modular organization of LMcryl which was found to encode a
mixed PKS/NRPS pathway. Three NRPS modules (1,2 and 3) precede a
single PKS module with an embedded TE (4) (B).
LMcryl encodes a large protein (-j 6500 amino acids) that contains three
NRPS modules followed by a PKS module which has an embedded thioesterase. The
first module possesses a Cy-A-PCP domain organization required for the activation
and heterocyclization of cysteine and is most similar to curacin CurF (51% identity)
(Chang et al., 2004) and barbamide BarG (50% identity) (Chang et al., 2002). The
condensation domain shows the presence of conserved motifs required for the
heterocyclization of cysteine when compared with the heterocyclization domains from
these two proteins (Figure 11.9). Analysis of the adenylation domain identified the
binding pocket amino acid residues that are in consensus for the activation of cysteine
(Table 11.3) (Challis et al., 2000). The absence of an embedded oxidase domain leads
71
us to speculate that heterocyclization results in the formation of a thiazoline ring as
opposed to a thiazole ring (Du et al., 2000; Schneider et al., 2003).
Cyl
Cy2
C3
Cy3
CYCCUrF
CycBarG
FPLNDIQQAYWIGR- - - -RMVVLADGNQQ- - - -DIFDAWS -- LPPAPEIP
FPLTEIQQAYWLGR- - - -RMVILPNGQQ- -- -DALII4DAWS - LPPAPELP
CycLMcryl FPLTDMQQAFWVGS- - - -RAVVLPDGQQK- - - -NPLILDTWS- - -FPESPELP
Cons.
FPLTxxQxAYxxGR----RxVxLPxGxQ ----- DxxxxDxxS-- --LPxxPExP
Cy4
Cy5
Cy6
Cy7
CycCurF
CycBarG
TPSGVLLSFASVLNYW- - -GDFTS- - - -GVVFTSTL- - -QVLLDHIVTEEKGALAFSWN
TPSGVLLAAFADAYW- - - -GDFTS - - - -GVVFTSTL- - -QVWLDNSVEQNGALLLIWH
CycLMcryl TPSG?LAAAFAEVLTRW- - - -GNFTS - - - -PVLFTSTL- - -RVWIDHQVYEENEALKFHWD
Cons.
TPxGxxxxxxxxVxxW- - - -GDFTS- - - -PVVFTSxL- - -QVxLDxQxxxxxxxxxxxWD
Figure 11.9: Alignment of heterocyclization domains. Barbamide BarG and
curacin A CurF aligned with heterocyclization domain from LMcryl show the
presence of consensus core motifs.
The second module consisted of a standard C-A-PCP structure and was found
to be most similar to module 2 in microcystin McyB (39% identity) (Tillett et al.,
2000). The adenylation domain was predicted to be specific for glutamine when
analyzed by the method of Challis et al (Table 11.3). However, comparison with other
cyanobacterial A-domains known to activate glutamine revealed considerable
differences in the binding pocket amino acids. An invariant Gln278 residue believed
to be involved in hydrogen bonding with substrate glutamine is replaced by Asn at this
position. However, this Asn residue could be involved in a similar interaction with the
substrate (Challis et al., 2000). We also compared the A-domain with asparagine
activating A-domain NosC3 from the nostopeptolide gene cluster (Hoffmann et al.,
2003) which revealed that the highly conserved G1u322 residue that is selective for
Asn was missing from this A-domain. Therefore, asparagine was eliminated as a
possible substrate (Stachelhaus et al., 1999). Similarly, conserved residues believed to
72
play an important role in the selection of aspartate (Thr239, His322) and glutamate
(Asp278) were also missing in this A-domain making it difficult to predict a putative
substrate for module 2. Although most binding pockets lend themselves easily to
chemical interpretation of their cognate amino acid substrate specificity, others clearly
do not. Additional biochemical characterization of this A-domain will be important in
determining its substrate specificity.
The presence of acidic amino acid residues in marine cyanobacterial
metabolites is relatively rare (Gerwick et al., 2001). This is in sharp contrast with
peptidic natural products of freshwater and terrestrial cyanobacteria, such as
microcystin (18) and nodularin (19), which contain acidic amino acid residues (Figure
11.11) (Carmichael et al., 1988; Krishnamurthy et al., 1986; Fujii et al., 1996).
Next, module three consists of a C-A-NM-PCP domain architecture and is
similar to module 2 in tubulysin TubC (37% identity) which also possesses identical
architecture (C-A-NM-A-PCP) (Sandmann et al., 2004). Analysis of the binding
pocket amino acids revealed that the A-domain was specific for valine and showed
good correlation with other valine-activating domains (Table 11.3). An N-methyl
transferase is embedded between the A9-A1 0 core motifs and shows the presence of
conserved residues and shows all the conserved residues when aligned with other N-
methyl tranferases from tubulysin TubC (Sandmann et al., 2004), microcystin McyA
(Tillett et al., 2000) and lyngbyatoxin LtxA (Edwards and Gerwick, 2004) (Figure
11.10).
73
Ml
M2
M3
N-MtLMcryl IVELGCGTG- -HNELNRFRYDV- - - IAYCRVPNARLMGEE
N-MtTubC
ILZVGCGTG- - -PNEMARERYNA---LGVAGIPNRVLPAV
N-MtMcyA
VLEIGCGTG- -HNELTQFRYNV- - -LRVIGVPNSRLFRPL
N-HtLtxPi
ILEIGVGTG- - -HNEMSQY?YNV- - -LKLVNVPNCRVTPAL
Figure 11.10: Alignment of N-methyl transferases from tubulysin
TubC, microcystin McyA, lyngbyatoxin LtxA with the LMcryl
N-MT shows the presence of conserved residues.
D-Gtu
D-GIu
COON
0
O
OCH3
CHJ
0-Me-Asp
COON
D-Me-Asp
Nodularin (19)
NI-I
HN
N H2
Microcystin (18)
Figure 11.11: Nonribosomal peptides from freshwater and terrestrial cyanobacteria.
Chemical structures show the presence of acidic amino acid residues such as
glutamate (Glu),and aspartate (Asp).
74
Table 11.3: Substrate specificities of A domain LMcryl and other related A-domains.
BarG (Chang et al., 2002), CurF (Chang et al., 2004), NcpA3 (Becker et al., 2004),
NosAl and NosC3 (Hoffmann et al., 2003), LtxAl (Edwards and Gerwick, 2004) and
TubC2 (Sandmann et al., 2004). The binding pocket amino acid residues correspond
to Grs-APhe A domain binding (Challis et al., 2000).
A-domain
Substrate
Binding pocket amino acids
235
236
239
278
299
301
322
330
LMcrylModAl
D
L
Y
N
I
G
I
V
cysteine
BarGc
D
L
Y
N
L
S
L
cysteine
CurFc
D
L
Y
N
V
D
L
I
I
LMcrylModA2
D
A
M
N
L
L
G
V
glutamine
NcpA3
D
A
W
Q
F
G
L
I
glutamine
ApdAl
D
A
W
Q
F
G
L
I
glutamine
NosC3
D
A
T
K
V
G
E
V
asparagine
LMcrylModA3
D
A
L
W
L
G
G
T
valine
NosAl
D
A
F
F
L
G
V
T
valine
LtxAl
D
I
Y
W
F
G
G
T
valine
TubC2
D
A
F
F
L
G
G
T
valine
cysteine
The PKS module reveals a KS-AT-CM-KR-ACP domain organization. The
incorporation of malonate versus methylmalonate extender units by a PKS is
determined by the AT-domain. In this case, the AT-domain in this module is
predicted to be specific for malonyl-CoA (Figure 11.12) (Haydock et al., 1995),
although a conserved valine is replaced by an isoleucine. The C-MT possesses a
signature motif (LExGxGxG) that is also present in the C-MT's from the jamaicamide
A and curacin A biosynthetic gene clusters (Figure 11.12) (Chang et al., 2004; Edwards
et al., 2004). An embedded type I thioesterase with the conserved GxSxG motif is
present at the C-terminal of this ORF and may likely catalyze the hydrolysis and
release of the product.
75
A
B
KS-JamK
KS-JamL
KS-CurF
KS-CurG
KS-LMcryl
Con.
AVDTACSSSL---YLB.AI3GT----GRTEGAA
AIDTACSASL---YVEAEGT---IGHLEAA
AVEAACSSSL---YLEABGT---XGNAATAA
TVQTACSTSL- - -YVEABGT- - -VGHLADP.A
AVQTACSTSL---YIETHGT---XGRLDAAA
DtaCS5SL
B
H
A-Jam1c
AT-JamL
AT-CUrF
AT-CurG
AT-LMcryl
Cons.
YTQPCLFLEYLCQL---MGHSVGE
YTQPALFAIEYALYKL---MGHSVGE
YTQPLFAIEThLWKL---MGHSVGE
YTQPM.FAIEYALFKL- - -NGHSVGE
FAQPALFAIEThLNQL---LGHSIGE
YTQ
E A
GHSV
(inalonyl C0A)
C-MT-JamJ
LLEIGAGTGGTT
C-14T-CurJ
LLEIAGTGGTT
C-MT-LMcryl ILEIGAGNGLLT
Cons.
ILHxGxGxGXxT
Figure 11.12: Sequence alignments of PKS domains of LMcryl with other
cyanobacterial PKS's (jamaicamide A and curacin A PKS genes). KS domain
alignment (A); AT-domain specific for malonyl-CoA (B); C-methyl transferase
domain alignment (C).
ORF II, is located about 700 bp downstream of LMcryl and encodes a putative
transposase. BLAST analysis of this gene showed that it was most similar to
transposase encoded by an 1S1136 type insertion sequence element from Clostridium
perfringens (33% identity) (Shimizu et al., 2002). Next, ORF III shows 52% identity
to a Natdriven multi-drug efflux pump from Nostoc punctiforme PCC73 102
(ZP_00 108972). The presence of these two proteins suggests that the TE domain in
the C-terminal end of LMcryl is the terminus of the gene cluster. Additional
sequencing will be required in order to determine the presence of other amino
acid/ketide incorporating domains or any tailoring enzymes that may be associated
with this gene cluster.
While sequence information upstream of ORF I remains to be determined, it
was apparent that this gene cluster was not involved in the biosynthesis of
hectochlorin (54). The presence of a module involved in the activation of an acidic
amino acid residue, an embedded N-methyl transferase in module 3 and the probable
termination of the cluster with a PKS segment eliminate its involvement in the
biosynthesis of hectochiorin (54). The gene cluster is most likely involved in the
biosynthesis of a metabolite whose deduced structure is represented in Figure 11.13.
,I1
NH2
Figure 11.13: Structure of proposed peptide encoded by hybrid NRPS/PKS
LMcryl. R unknown side chain.
A curious insight resulting from sequence analysis of cosmid 1-105 is the
presence of short regions dispersed over an 8 kb fragment that show similarity to
cyanobacterial transposases such as those from Nostoc punctforme PCC73 102
(ZP_00 106214), Anabaena variabilis ATCC294 13 (ZP_00 162592) and Crocosphaera
watsonii (ZP_00 1779422). The presence of such interdigitated putative transposases
has also been reported in Synechocystis PCC6803 (Mahillon et al., 1999). These may
have resulted from the insertion of several IS elements that may be rendered inactive
over time as a result of mutations (Meeks et al., 2001).
Our strategy which involved the use of general adenylation domain probes
proved to be unsuccessful in isolating the hectochlorin biosynthetic gene cluster. The
homology-based approach (Turgay and Marahiel, 1994) was not feasible for
organisms known to harbor multiple PKS/NRPS gene clusters in their genomes and
lately there has been a shift towards the use of highly specific approaches to isolate
secondary metabolite gene clusters from organisms known to possess multiple
PKS/NRPS gene (Cheng et al., 2002; Sandmann et al., 2004; Edwards et al., 2004).
Sequence analyses of several bacterial genomes have revealed that there is
often discrepancy between the number of secondary metabolite gene clusters harbored
in the genome of an organism and the number of natural products that can be isolated.
77
For example, genome sequencing of Streptomyces avermitilis revealed the presence of
twenty five secondary metabolite gene clusters that accounted for 6.4% of the total
genome size and putative products encoded by five PKS and eight NRPS clusters have
not yet been identified (Omura et al., 2001). Similarly, Streptomyces coelicolor shows
the presence of about twenty gene clusters involved in the biosynthesis of secondary
metabolites, but the corresponding metabolites formed from several of these remain
unidentified (Bentley et al., 2002). Mxyobacteria such as Stigmatella aurantiaca and
Sorangium cellulosum So ce9O have also been shown to harbor multiple hybrid
PKS/NRPS gene clusters (Pradella et al., 2002; Silakowski et al., 2001).
Cyanobacteria are emerging as prolific producers of secondary metabolites and
filamentous cyanobacteria such as Nostoc punctforme, Anabaena sp, Lyngbya
majuscula tend to harbor a number of gene clusters involved in secondary metabolite
bioynthesis. Analysis of the recently annotated genome of the terrestrial
cyanobacterium, Nostocpunctforme has revealed the presence of at least 16
secondary metabolite gene clusters and for many of these, a corresponding secondary
metabolite has not yet been isolated (Salomon et al., 2004). The genome of
filamentous cyanobacterium Anabaena PCC 7120 also shows the presence of several
biosynthetic gene clusters involved in the production of natural products of
PKS/NRPS origin (http://www.kazusa.or.jp/cyano/Anabaenalindex.html). While
genome sequence data for Lyngbya majuscula is currently unavailable, strains like L.
majuscula 19L have been shown to possess multiple gene clusters of which three are
completely characterized: barbamide (Chang et al., 2002) curacin (Chang et al., 2004)
and carmabin (Chang et al., unpublished). Re-screening of a L. majuscula JRB
genomic library concurrent to this work also resulted in the identification of multiple
PKS/NRPS gene clusters (D.J. Edwards, unpublished).
Among organisms that possess multiple PKS/NRPS gene clusters, a critical
question that is often arises is whether the organism expresses all of these clusters to
produce secondary metabolites under normal culture conditions. In the case of marine
cyanobacteria whose natural habitat is a complex ecosystem, a delicate balance of
physical and nutrient conditions along with other habitat interactions influence growth
I3
and production of secondary metabolites (Patterson et al., 1994). The fact that certain
cyanobacterial strains are more amenable to laboratory cultivation does not necessarily
mean that they retain all of their biosynthetic capabilities to produce secondary
metabolites. Indeed, frequent loss, alteration or decreased metabolite production is
encountered in cultured cyanobacteria (Moore et al., 1988).
Nevertheless, several groups have succeeded in the isolation of numerous
bioactive metabolites from cultured cyanobacteria (Gerwick et al., 1994; Patterson et
al., 1991; Patterson et al., 1993; Patterson et al., 1994; Rossi et al., 1997; Schwartz et
al., 1990). A Caribbean collection of L. majuscula which produces the antitubulin
agent, curacin A, was successfully adapted to laboratory culture. Furthermore, even
after extended culturing, production of curacin A in the cultured strain was
comparable to that found in wild material (Rossi et al., 1997). Intensive culture efforts
have also led to the identification of an axenic strain of the cyanobacterium,
Scytonema ocellatum, which produces twice as much tolytoxin as the parental strain
(Patterson et al., 1994).
Therefore, it is interesting to establish whether the putative metabolite encoded
by the cryptic cluster, LMcryl is produced by cultures L. majuscula JHB. This
prompted us to re-examine the organism's secondary metabolite profile. The crude
organic extract (CH2Cl2-MeOH, 2:1) of the cultured organism was fractionated using a
standard VLC protocol (Marquez et al., 2002) to give nine fractions (A-I). Reversed
phase HPLC profiling of these fractions revealed three major components that were
present in large amounts (Figure 11.14) and corresponded to four previously
characterized metabolites, hectochiorin (54) and jamaicamides A-C (27-29)(Edwards
et al., 2004; Marquez et al., 2002). Although fractions B, C and E contained several
small peaks, these proved to be in sub-milligram quantities (<0.2 mg) of mostly
impure materials. Consequently, no structure characterization by NMR or other
spectral methods was attempted (K. L. McPhail, unpublished).
Jamaicamide A (27)
Hectochlorin (54)
Jamaicamide B (28)
A
A
A
.1
ii
-
ft
Jamaicamide C (29)
crude
________________
3
.1
____
Hectochlorin (54)
)
Jamaicamide A (27)
Jamaicamide B (28)
AU
m:mideC(29)
Hectochlorin (54)
Jamaicamide A (27)
Jamaicamide B (28)
jrnidec(29)
Jamaicamide A (27)
Jamaicamide B (28)
:boni54
JJamaicamide C (29)
-I
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.0
Minutes
Figure 11.14: Reversed phase HPLC profiles for the crude extract and nine normal
phase VLC fractions (A-I) of the cultured Lyngbya majuscula JHB. In each case
10 pL (0.25 mg) of sample was injected onto an analytical colunm (Varian
Microsorb-MV 100-5 C18) and eluted over 50 mm, firstly with a gradient of 50%
acetonitrile/water through 100% acetonitrile (40 mm) and then continued elution
with 100 % acetonitrile (10 mm). All samples were monitored at the same 5
wavelengths (211, 230, 254, 270, 320 nm) using a Waters 996 Photodiode Array
detector. Results are plotted at exactly the same vertical scale in each case.
80
Therefore, because the crude extract of L. majuscula JHB contained several
candidate metabolites of limited mass, we decided to explore a more targeted and
novel strategy that combined aspects of molecular biology and natural products
chemistry. The first step in this approach is demonstration of LMcryl expression
using reverse transcriptase-PCR (RT-PCR) or Northern blot analysis. In the next step,
bioinformatics prediction of the adenylation domain specificities is used to select
doubly labeled 13C - 15N amino acid precursors that are be fed to a culture of L.
majuscula JHB. Finally, selective 13C-'5N 2D NMR methods, such as the gradient
hydrogennitrogen multiple quantum coherence (GHNMQC) experiment, will be used
in an "NMR-guided" fractionation of the labeled extract to isolate the putative
metabolite, which can then be characterized by routine 2D NMR spectroscopy and
mass spectrometry (Figure 11.15).
Isolation of cryptic gene cluster
Demonstration of gene
expression
[
(
J
Bioinformatics prediction of
A-domain specificities/
Experimenatal determination of
Lsubstrate specificity
Labeled amino acid precursor feeding experiments
based on A-domain specificity
rIsolation and structural elucidation of corresponding
bmetabolite using select 2D-NMR methods
Figure 11.15: Schematic representation of the novel approach used to isolate
and elucidate structure of putative metabolite encoded by LMcryl.
81
Demonstration of LMcryl expression
A number of investigations have been carried out to study the influence of
environmental factors on secondary metabolite production in cyanobacteria (Patterson
and Bolis, 1995; Rapala et al., 1997; Repka et al., 2004; Watanabe and Oishi, 1985).
However, there is very little understanding of cyanobacterial secondary metabolite
regulation at the genetic level and thus far, expression analysis has been reported only
for the microcystin biosynthetic gene cluster (Kaebernick et al., 2000; Kaebernick et
al., 2002).
Various methods are available for the detection and monitoring of bacterial
gene expression including Northern hybridization, RNase protection assay and
different forms of RT-PCR. Northern hybridization has a major advantage in that it is
the only method which provides information regarding the size of the transcript
(Sharkey et al., 2004). However, Northern blot analyses could not detect expression
of the microcystin gene cluster. Low transcript levels and potential length of the
transcripts were believed to be responsible for this observation (Kaebernick et al.,
2002). Therefore, we decided to use the RT-PCR approach. The first step in RT-PCR
is the production of cDNA from mRNA using retroviral reverse transcriptase. The
cDNA then serves as a template for exponential amplification by PCR. RT-PCR has
become an indispensable tool for examining gene expression and can even be used to
detect transcripts present in very low abundance.
Specific primers were designed to amplify approximately 500 bp regions along
the length of the LMcryl as follows: primers F 1 fRi amplified a region in the first
module, F2/R2 amplified a region within the second module and F3/R3 were designed
to amplify a small region at the end of module three (Figure 11.16). A small region of
the jamB gene from jamaicamide A biosynthetic gene cluster (Edwards et al., 2004)
was selected as a positive control and specific primers for its amplification were also
designed.
82
Fl/Ri
I
Modei
F2/R2
I
Module 2
F3/R3
I
Module 3
I
Module 4
LMciyl
Figure 11.16: Primers for RT-PCR. Primers Fl/Ri amplified a 520 bp
region within module 1, primers F2/R2 amplified a 500 bp region
within module 2 and F3/R3 amplified a 500 bp region towards the end
of module 3.
Total RNA was isolated from cultures of L. majuscula JHB using RNAwiz
(Ambion) at early (2-week old culture) and late (5-week old culture) logarithmic
growth stages. The isolated total RNA was treated with DNase to remove any
contaminating DNA before it was used as template for RT-PCR. Concentration and
purity were determined using a spectrophotometer. Generally, RT-PCR can be
performed in a one-step format or two-step format. In the former, reverse
transcription and PCR take place in a single tube which reduces the risk of carryover
contamination, whereas in the latter format, each step is performed in separate tubes
and 10% of the reverse transcription reaction is used as template for PCR (Sharkey et
al., 2004). Initially, we used the one-step format but this method proved unreliable
and failed to produce consistent results. We then tested the two-step format which
was highly successful and could be replicated readily. Total RNA was converted to
cDNA using the gene-specific reverse primers, Ri, R2, R3 and jamB-R, and cDNA
obtained was used as template for subsequent PCR reactions. Amplicons were
obtained for Fl/Ri, F2/R2 from cultures in both early and late logarithmic growth
stages, thus demonstrating expression of the LMcryl in the cultured L. majuscula JHB
(Figure 11.17). The presence of transcripts in the early and late logarithmic cultures
was also consistent with the observation that cyanobacteria, unlike Streptomyces sp.,
appear to produce secondary metabolites throughout the growth-phase (Orr and Jones,
1998; Repka et al., 2004; Rossi et al., 1997). However, primer pair F3/R3 failed to
amplify the corresponding fragment from both culture samples. A likely explanation
for this observation may be the location of these two primers at the 3'-end of the
transcript which is more susceptible to degradation by RNases (Kennell, 2002;
Kushner, 2002; Steege, 2000). Amplification ofjamB positive control from both
cultures was also successful. Negative control experiments in which RNA was used
as template for PCR did not result in amplification, thus indicating the complete
absence of contaminating DNA in the total RNA sample (Figure 11.17). All PCR
products were subcloned into pGEM-T Easy vector and their sequences were verified
as deriving from the LMcry 1 cluster.
DNA template
Marker
p.
Fl/Ri F2/R2 F3/R3 Neg
con.
Pos
con.
4 DNA template 0
Marker
Fl/Ri F2/R2 F31R3 Neg Pos
con. con.
* RNA template 0
Fl/Ri F2/R2 F31R3 Neg
con.
Pos
con.
4 RNA template +
Fl/Ri F2/R2 F31R3 Neg Pos
con. con.
Figure 11.17: Results of RT-PCR. RT-PCR analysis using primers Fl/Ri,
F2/R2, F31R3 and jamaicamide jamB as positive control confirms expression
of LMcryl characterized from cultures of L. majuscula JHB in early (A) and
late (B) logarithmic phases. Negative control experiments in which RNA was
used as template revealed complete absence of contaminating DNA.
Thus expression of LMcryl in cultured L.
majuscula
JHB was successfully
demonstrated using RT-PCR. Precursor feeding studies with labeled amino acid are
currently underway and will hopefully lead to the identification of the corresponding
peptide natural product.
With the identification and characterization of over 150 hybrid PKSINRPS
gene clusters from several organisms (Salomon et al., 2004), our understanding of
these systems has advanced to the point where we can use sequence information to
assign structures with reasonable accuracy. Natural products continue to be a major
source of molecules for drug discovery; however, development of new methodologies
is vital for the identification of novel chemical structures and methods for their
production. As the number of gene clusters encoding unknown PKS/NRPS
metabolites increases with the rapidly accumulating genome sequence data from
several organisms, the utility of genomics in conjunction with standard spectroscopic
techniques will become invaluable for the isolation and structural elucidation of novel
metabolites.
EXPERIMENTAL
General
All standard DNA manipulations such as restriction digests, ligations and
transformations were carried out using standard methods (Sambrook Ct al., 1989).
Restriction digest enzymes were purchased from several vendors and used according
to manufacturer's recommendations.
Bacterial strains and culture conditions
Escherichia coli strain DH1OB was used as a host for DNA cloning and
sequencing, while E. coli EPI 100 was used as a vector for construction of cosmid
library. E. coli DH1OB cells harboring pGEM-T Easy, pGEM3ZF(+) or pBluescript
was grown at 37°C overnight in Luria-Bertani medium (LB) with a final concentration
of ampicillin at 100 tg/mL. E. coli EPI 100 cells with cosmid pWEB::TNC were
grown at 37°C in LB medium supplemented with 100 jtg/mL ampicillin.
Lyngbya majuscula strain JHB, Lyngbya majuscula strain 19L and Phormidium sp
were grown under previously described conditions (Marquez et al., 2002; Rossi et al.,
1997; Williamson et al., 2002). Genomic DNA for library construction was isolated
from a four-week old culture of Lyngbya majuscula JHB. Lyngbya MNT 01 was
collected near Nosy Tanikely, Madagascar (April
17th
2000) and Lyngbya NPI 03 was
collected in Curacao (May 11th 1996) and preserved in isopropanol/seawater mixture
(60:40). Both preserved samples were stored at -20°C.
Isolation of genomic DNA
Method I (Asper.-illus prep): About one gram of each cyanobacteria was ground in
liquid nitrogen and treated with 0.6 mL of extraction buffer [0.1 M Na2(S03) , 0.1 M
Tris-Cl pH 7.5, 0.05 EDTA, and 1% (w/v) SDS} and incubated at 65°C for 20
minutes. The lysate was then treated with an equal volume of chloroform-isoamyl
alcohol and incubated on ice for 30 minutes. After centrifugation at 12000 rpm for 30
minutes, the aqueous layer was transferred to a fresh tube and genomic DNA was
precipitated by the addition of isopropanol. It was then washed 2x times with 70%
ethanol and resuspended in 50 p.L water and incubated at 4°C to aid resuspension.
Method II (CTAB buffer): About one gram of cyanobacteria was ground to a fine
powder using liquid nitrogen and treated with 5 mL of TE25S buffer (0.025 M TrisHC1 [pH 8], 0.025 M EDTA [pH 8], 0.3 M sucrose) with 2 mg!mL lysozyme and
incubated for one hour at 37°C. Proteinase K and SDS at a final concentration of 0.18
mg!mL and 0.5%, respectively were then added and incubated for 1 hour at 55°C.
Next, the lysate was treated with 0.5 M NaCl and extracted twice with 10% CTAB
(hydroxyacetyl trimethyl ammonium bromide)! 0.7 M NaCI followed by 24:1
chloroform: isoamyl alcohol. Genomic DNA was finally precipitated with
isopropanol and washed twice with 70% ethanol.
Method III : Two grams (wet weight) of L. majuscula JHB, L. majuscula 19L or
Phormidium sp was harvested and rinsed three times with 20 mL of sterile Instant
Ocean BG1 1 medium, followed by 20 mL of 10 mM Tris (p11=8) in a Buchner funnel.
The tissue was then flash-frozen in liquid nitrogen and homogenized in a pre-chilled
mortar and pestle using liquid nitrogen. After evaporation of excess liquid nitrogen,
the resulting fine powder was resuspended in 20 mL of lysis buffer (10 mM Tris
[pH=8], 0.1 mM EDTA [pH=8J, 0.5% (w/v) SDS and 20 .tg/mL DNase free RNase A
and incubated for 1 hour at 37°C. During incubation, the lysate was gently mixed by
inversion every 20 minutes. Proteinase K (20 mg/mL) was added to the lysate at a
final concentration of 100 p.g!mL and incubated at 50°C for 1 hour. The solution was
cooled to room temperature before adding 20 mL of phenol equilibrated with 0.1 M
Tris [pH=8]. The two phases were gently mixed for 10 minutes on a platform shaker
and the phases were separated by centrifugation at 25°C for 10 minutes at 4000 rpm.
The viscous aqueous phase was transferred by careful pipetting to a clean tube using a
wide-bore tip (0.3 cm orifice diameter). The extraction with phenol was repeated two
more times and the aqueous extracts were pooled. The pooled aqueous extracts were
treated with 0.2 volumes of 10 M ammonium acetate and 2 volumes of absolute
ethanol at room temperature. After gentle mixing, the DNA was removed from the
aqueous extract by using a sterile Pasteur pipette and transferred to a fresh tube. The
DNA was then washed with 70 % ethanol three times and re-suspended in 500 .t1 of
low TE (2 mM Tris [pH=8] and 0.2 mM EDTA) and incubated at 4°C for 12-24 hours.
After re-suspension genomic DNA was subjected to further purification using
Qiagen's Genomic Tip 100 according to instructions provided with the kit.
Concentration and purity (A260/A280) of genomic DNA were measured using a
Beckman DU600 spectrophotometer.
Genomic DNA was isolated using DNeasy Plant Mini Kit (Qiagen), DNAzo1
(Gibco Life Sciences) and Wizard DNA Purification Kit (Promega) according
manufacturer's protocols. The DNeasy kit was used for the isolation of genomic DNA
for PCR amplification of probes.
Amplification of PKS and NRPS probes
PCR was carried out using Eppendorf Mastercycler in 50 j.tL reaction volumes
containing 40 ng of genomic DNA template, 5 tL of PCR buffer (Invitrogen), 50 mM
MgC12, 10 mM dNTP's, 10 jtM primers and 2.5 units of Taq DNA polymerase
(Invitrogen). Degenerate primers KS1Up 5' MGI GAR GCI HWI SMI ATG GAY
CCI CAR CAT MG 3' and KSD1 5' GGR TCI CCI ARI SWI GTI CCR TG 3' were
used for the amplification of KS domains from PKS (Beyer et al., 1999). Primers
A2Forward 5' GCN GGY GGYGCN TAY GTN CC 3' and A8Reverse 5' CCN CGD
ATY TTN ACY TG 3' were used for the amplification of NRPS adenylation domain
probes (Neilan et al., 1999). The following thermocycler conditions were used for
amplification: denaturation at 94°C for 30 s, annealing at 48°C for 30 s, extension at
72°C for 1 mm for 30 cycles. The amplification of cysteine-specific adenylation
domain primers were conducted using primers, 720F 5' TTY GAY YTI WSI GTI
TAY GAY ITI TTY GG 3' and 840R 5' TGR TTI CCI ART CSI CYI CCI TAI GGI
AT 3' using the following thermocycler conditions: denaturation at 94°C for 5 mins,
annealing at 45°C for 30 s, extension at 72°C for 1 mm for 30 cycles. All primers
were obtained from Invitrogen (Carlsbad).
The PCR products were purified using Qiagen's PCR purification kit and
cloned into pGEM-T Easy for sequencing. Unique NRPS clones were identified by
digestion with TaqI for 1 hour and representative clones from each group were
sequenced. Sequencing reactions were performed at the Central Services Lab, Center
for Gene Research and Biotechnology at Oregon State University and Davis
Sequencing (Davis, California). Sequence analysis was carried out using National
Center for Biotechnology Information's (NCBI) BLAST tool (Altschul et al., 1990)
and ClustalW (Thompson et al., 1994).
Construction of cosmid library
High molecular weight genomic DNA (-.. 40 kb) was end-repaired and size
selected by electrophoresis on a 0.7% low-melt agarose gel. The gel slice was
equilibrated in 5 volumes of Gelase buffer three times and subjected to overnight
digest with 5 units of Gelase®. Library construction was conducted using Epicentre's
pWEB::TNC cosmid cloning kit. Purified genomic DNA was then ligated into
pWEB::TNC vector and packaged into MaxPlax phage extracts according to
instructions accompanying the kit. The packaged phage was then used to infect E. coli
EPI 100 cells and an average titer of 2.2 x
io
cfulmL was obtained.
Screening of library
Library clones were inoculated in a numbered grid on 150 mm Petri dishes
containing LB agar with 100 jtg/mL ampicillin. All clones were inoculated in
duplicates. Colonies were transferred to nylon membranes (Hybond N, Amersham
Pharmaecia Biosciences) and were processed for colony hybridization using standard
procedures (Sambrook et al., 1989). The DNA was fixed to the nylon membrane by
cross-linking in a CL1 000 Ultraviolet Crosslinker.
The amplified PKS and NRPS probes were purified prior to labeling. Probes
were labeled with dioxygenin using the DIG High Prime DNA Labeling and Detection
Starter Kit II (Roche Molecular Biosciences) and 40 ng/mL of labeled probe was used
for all colony hybridizations. Colony hybridization was carried out according to
instructions provided with the DIG High Prime Kit. Prehybridization of nylon
membranes was carried out at 42°C, hybridization at 42°C and stringency washes
were performed at 50°C. The addition of CSPD (Disodium 3-(4-meth-oxyspiro {l,2-
dioxetane-3,2'-(5'-chloro) tricyclo [3.3.l.l'J decan}-4-yl) phenyl phosphate) provided
with the kit serves as a substrate for the detection of DIG labeled positive colonies by
chemiluminescence.
Southern analysis
Purified cosmid DNA ('-1 pjg) was digested with HindIlI, EcoRI, BamHI, PstI
and NcoI and separated on a 1% agarose gel. After pre-treatment of gel according to
standard protocols (Sambrook et al., 1989), prehybridization, hybridization, washing
were performed as described for the colony lifts. Labeled probes were used at a
concentration of 20 ng/mL. Detection was also carried out as described for colony
hybridizations.
Sub-cloning of cosmid 1-105
Cosmid DNA (-
1
tg) was dissolved in 300 i.tL of deionized water for
sonication using XL2000 MicrosonTM Ultrasound homogenizer at 100W for 20 s with
a 1/8" probe. The fragmented DNA was end-repaired using End-It® from Epicentre
Technologies and separated on a 1% gel. Fragments within the desired size range
(1.5-2 kb) were purified and ligated into prepared pGEM3Zf(+) vector.
Cosmid DNA (-1 jig) was subjected to partial digest using A1uI or Sau3AI
(0.15-0.20 units/jig of cosmid DNA for 1 hour). The partially digested cosmid DNA
was then separated on a 1% agarose gel and fragments of size 1-2.5 kb were selected.
After gel purification, the A/uI fragments were ligated into pGEM3Zf(+) vector treated
with SmaIICTP (Calf Intestinal Phosphatase). Similarly, Sau3Al fragments were
ligared into pBluescript that was digested with BamHI /CIP. Random sub-clones were
selected for sequencing. Sequencing was carried out using Big Dye III terminator
cycle sequencing (PE Biosystems) at the Central Services Lab, Center for Gene
Research and Biotechnology, Oregon State University. Resulting gaps were filled
using primer walking with sequence specific primers. The sequences were edited and
assembled using Vector NTI's Contig Assembly software program (InforMax,
Bethesda, MD). Analyses of DNA and protein sequences were carried out using the
NCBI's BLAST server(Altschul et al., 1990). All alignments were conducted using
ClustalW (Thompson et al., 1994).
Isolation of RNA-Gen era!
Prior to isolation of total RNA, all equipment and accessories were
meticulously cleaned to eliminate any contaminating RNases. Microfuge tubes and
pipette tips were autoclaved for 30 minutes and all glassware was autoclaved twice for
30 minute each. Pipettes, gel apparatus, microcentrifuge and general work area were
wiped with RNase Zap (Ambion, Austin,TX). During the RNA isolation process,
gloves were periodically changed, in order to prevent any carry-over contamination
resulting from contact with gloves. Water was treated with 1% (v/v) DEPC for 18
hours and autoclaved for 30 minutes. All reagents were made in DEPC-treated water
and autoclaved before use.
Isolation of total RNA from L.
majuscula
JHB
For isolation of total RNA L. majuscula JHB was grown under previously
described conditions (Marquez et al., 2002). Total RNA was isolated using RNAwiz
(Ambion, Autin, TX). About 1.82 g (wet weight) of a 2-week old culture and 2.08 g
(wet weight) of a 5-week old culture was harvested and homogenized in liquid
nitrogen using a mortar and pestle. The finely ground tissue was resuspended in an
appropriate volume of RNAwiz and isolation of RNA were carried out according to
manufacturer's protocol. Total RNA was resuspended in 50 jiL of DEPC-treated
water. In order to ensure complete removal of DNA, the isolated total RNA was
treated with 1 iL of Turbo DNase (Ambion, Austin,TX) for 30 minutes at 37 °C. The
DNase was inactivated by the addition of DNase Inactivation reagent and incubating
for 2 minutes at room temperature. Total RNA obtained by this method was used for
the generation of cDNA. Total RNA was quantified using a Beckman DU600
spectrophotometer
91
Generation of cDNA and RT-PCR
Reverse transcription of total RNA to generate cDNA was performed using
instructions accompanying SuperScript TM JJJ First-Strand Synthesis System
(Invitrogen, Carlsbad, CA) for RT-PCR. Briefly, 10 1.tL of total RNA template was
incubated with 1 tL of gene-specific reverse primer (10 j.M), 1 xL dNTP, appropriate
buffers and 1 tL of SS-Reverse transcriptase for 1 hour at 55°C. After inactivation of
the reverse transcriptase, cDNA was used as a template for RT-PCR.
Primers used for RT-PCR were as follows: F 1-5' GCA AAA GGC TAT TGG
CGC 3' and R1-5' GTT CCT CAA TGG AAA TGC G 3'; F2-5' GTT GTA GAT
TGG AAT CAG 3' and R2-5' GGT CAG AAC CAC GCC TTT 3'; F3-5' GAA CTG
TAT GTA GCA GGA GA 3' and R3-5' TCC CAA TTC CAC GAT GCG 3'; jamB-F
5' ATG TCA ATG CCA ATG GAT GT 3' and jamB-R 5' GGT CTT TAC TAT CGA
AAG GGT 3'. The following PCR conditions were used: denaturation at 94°C for 1
mi
annealing at 55°C for 45 s and extension at 72°C for 1 mm for 30 cycles. PCR
products were purified using Qiagen's PCR purification kit and cloned into pGEM-T
Easy vector for sequencing.
92
REFERENCES
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic
local alignment search tool. Journal of Molecular Biology 215, 403-410.
Becker, J. E., Moore, R. E., and Moore, B. S. (2004). Cloning,sequencing,and
biochemical characterization of the nostocyclopeptide biosynthetic gene
cluster:molecular basis for imine macrocyclization. Gene 325, 3 5-42.
Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M., Challis, G. L., Thomson, N. R.,
James, K. D., Harris, D. E., Quail, M. A., Kieser, H., Harper, D., Bateman, A., Brown,
S., Chandra, G., Chen, C. W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo,
J., Hornsby, T., Howarth, S., Huang, C. H., Kieser, T., Larke, L., Murphy, L., Oliver,
K., O'Neil, S., Rabbinowitsch, E., Rajandream, M. A., Rutherford, K., Rutter, S.,
Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T.,
Wietzorrek, A., Woodward, J., Barrell, B. G., Parkhill, J., and Hopwood, D. A. (2002).
Complete genome sequence of the model actinomycete Streptomyces coelicolor
A3(2). Nature 417, 141-7.
Beyer, S., Kunze, B., Silakowski, B., and Muller, R. (1999). Metabolic diversity in
myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene
cluster of Stigmatella aurantiaca Sg a15 and a combined polyketide-(poly)peptide
gene cluster from the epothilone producing strain Sorangium cellulosum So ce9O.
Biochimica Et Biophysica Acta 1445, 185-195.
Bierman, M., Logan, R., O'Brien, K., Seno, E. T., Rao, R. N., and Schoner, B. E.
(1992). Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia
coli to Streptomyces spp. Gene 116, 43-49.
Carmichael, W. W., Eschedor, J. T., Patterson, G. M., and Moore, R. E. (1988).
Toxicity and partial structure of a hepatotoxic peptide produced by the
cyanobacterium Nodularia spumigena Mertens emend. L575 from New Zealand.
Applied and Environmental Microbiology 54, 225 7-2263.
Challis, G. L. and Ravel, J. (2000). Coelichelin, a new peptide siderophore encoded by
the Streptomyces coelicolor genome: structure prediction from the sequence of its nonribosomal peptide synthetase. FEMS Microbiology Letters 187, 111-114.
Challis, G. L., Ravel, J., and Townsend, C. A. (2000). Predictive, structure-based
model of amino acid recognition by nonribosomal peptide synthetase adenylation
domains. Chemistry & Biology 7, 211-224.
Chang, Z., Flatt, P., Gerwick, W. H., Nguyen, V. A., Willis, C. L., and Sherman, D. H.
(2002). The barbamide biosynthetic gene cluster: a novel marine cyanobacterial
system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS)
origin involving an unusual trichloroleucyl starter unit. Gene 296, 235-247.
Chang, Z., Sitachitta, N., Rossi, J. V., Roberts, M. A., Flatt, P. M., Jia, J., Sherman, D.
H., and Gerwick, W. H. (2004). Biosynthetic pathway and gene cluster analysis of
curacin A, an antitubulin natural product from the tropical marine cyanobacterium
Lyngbya majuscula. Journal of Natural Products 67, 1356-1367.
Cheng, Y. Q., Tang, G. L., and Shen, B. (2002). Identification and localization of the
gene cluster encoding biosynthesis of the antitumor macrolactam leinamycin in
Streptomyces atroolivaceus S-140. Journal of Bacteriology 184, 7013-7024.
Du, L., Chen, M., Sanchez, C., and Shen, B. (2000). An oxidation domain in the
Bimill non-ribosomal peptide synthetase probably catalyzing thiazole formation in the
biosynthesis of the anti- tumor drug bleomycin in Streptomyces verticillus
ATCC15003. FEMS Microbiology Letters 189, 171-175.
Edwards, D. J. and Gerwick, W. H. (2004). Lyngbyatoxin biosynthesis: sequence of
biosynthetic gene cluster and identification of a novel aromatic prenyltransferase.
Journal of American Chemical Society 126, 11432-11433.
Edwards, D. J., Marquez, B. L., Nogle, L. M., McPhail, K., Goeger, D. B., Roberts, M.
A., and Gerwick, W. H. (2004). Structure and biosynthesis of the jamaicamides, new
mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya
majuscula. Chemistry & Biology 11, 8 17-833.
Fiore, M. F., Moon, D. H., Tsai, S. M., Hung, L., and Trevors, J. T. (2000). Miniprep
DNA isolation from unicellular and filamentous cyanobacteria. Journal of
Microbiological Methods 39, 159-169.
Fujii,K., Harada,K.-I., Suzuki,M., Kondo,F., Ikai,Y., Oka,H., and et al. (1996).
Occurence of novel cyclic depsipeptides togteher with microcystins from toxic
cyanobacteria Anabaena species. Tn Harmful and Toxic Algal Blooms, T.Yasumoto,
Y.Oshima, and Y.Fukuyo, eds. (Paris: Intergovernmental Oceanographic Commission
of UNESCO), pp. 559-562.
Gehring, A. M., Mori, I., Perry, R. D., and Walsh, C. T. (1998). The nonribosomal
peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of
yersiniabactin, an iron-chelating virulence factor of Yersinia pestis. Biochemistry 37,
11637-11650.
Gerwick, W. H., Roberts, M. A., Proteau, P. J., and Chen, J.-L. (1994). Screening
cultured marine microalgae for anticancer-type activity. Journal of Applied Phycology
6, 143-149.
Gerwick,W.H., Tan,L., and Sitachitta,N. (2001). Nitrogen-containing metabolites from
marine cyanobacteria. In The Alkaloids, G.A.Cordell, ed. (San Diego: Academic
Press), pp. 75-184.
94
Haydock, S. F., Aparicio, J. F., Molnar, I., Schwecke, T., Khaw, L. E., Konig, A.,
Marsden, A. F., Galloway, I. S., Staunton, J., and Leadlay, P. F. (1995). Divergent
sequence motifs correlated with the substrate specificity of (methyl)malonyl-00A:acyl
carrier protein transacylase domains in modular polyketide synthases. Febs Letters
374, 246-248.
Hofthiann, D., Hevel, J. M., Moore, R. B., and Moore, B. S. (2003). Sequence analysis
and biochemical characterization of the nostopeptolide A biosynthetic gene cluster
from Nostoc sp. GSV224. Gene 311, 171-180.
Kaebernick, M., Dittmann, E., Borner, 1., and Neilan, B. A. (2002). Multiple alternate
transcripts direct the biosynthesis of microcystin, a cyanobacterial nonribosomal
peptide. Applied and Environmental Microbiology 68, 449-55.
Kaebernick, M., Neilan, B. A., Borner, T., and Dittmann, B. (2000). Light and the
transcriptional response of the microcystin biosynthesis gene cluster [In Process
Citation]. Applied and Environmental Microbiology 66, 3387-92.
Kaneko, T., Nakamura, Y., Wolk, C. P., Kuritz, T., Sasamoto, S., Watanabe, A.,
Triguchi, M., Ishikawa, A., Kawashima, K., Kimura, T., Kishida, Y., Kohara, M.,
Matsumoto, M., Matsuno, A., Muraki, A., Nakazaki, N., Shimpo, S., Sugimoto, M.,
Takazawa, M., Yamada, M., Yasuda, M., and Tabata, S. (2001). Complete genomic
sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC
7120. DNA Research 8, 205-213.
Keimell, D. (2002). Processing endoribonulceases and mRNA degradation in bacteria.
Journal of Bacteriology 184, 4645-4657.
Konz, D., Klens, A., Schorgendorfer, K., and Marahiel, M. A. (1997). The bacitracin
biosynthesis operon of Bacillus lichenformis ATCC 10716: molecular
characterization of three multi-modular peptide synthetases. Chemistry & Biology 4,
927-937.
Krishnamurthy, T., Carmichael, W. W., and Sarver, B. W. (1986). Toxic peptides from
freshwater cyanobacteria (blue-green algae). I. Isolation, purification and
characterization of peptides from Microcystis aeruginosa and Anabaena flos-aquae.
Toxicon 24, 865-873.
Kushner, S. R. (2002). mRNA decay in Escherichia coli comes of age. Journal of
Bacteriology 184, 4658-4665.
Mahillon, J., Leonard, C., and Chandler, M. (1999). IS elements as constituents of
bacterial genomes. Research in Microbiology 150, 675-687.
Marquez, B. L., Watts, K. S., Yokochi, A., Roberts, M. A., Verdier-Pinard, P.,
Jimenez, J. I., Hamel, E., Scheuer, P. J., and Gerwick, W. H. (2002). Structure and
absolute stereochemistry of hectochiorin, a potent stimulator of actin assembly.
Journal of Natural Products 65, 866-87 1.
McAlpine, J. B., Bachmann, B. 0., Piraee, M., Tremblay, S., Alarco, A.-M.,
Zazopoulos, E., and Farnet, C. M. (2005). Mircobial genomics as a guide to drug
discovery and structural elucidation: ECO-02301, a novel antifungal agent, as an
example. Journal of Natural Products 68, 493-496.
Meeks, J. C., Elhai, J., Thiel, T., Potts, M., Larimer, F., Lamerdin, J., Predki, P., and
Atlas, R. (2001). An overview of the genome of Nostocpunctforme, a multicellular
symbiotic cyanobacterium. Photosynthesis Research 70, 85-106.
Molnar, I., Schupp, T., Ono, M., Zirkie, R., Milnamow, M., Nowak-Thompson, B.,
Engel, N., Toupet, C., Stratmann, A., Cyr, D. D., Gorlach, J., Mayo, J. M., Hu, A.,
Goff, S., Schmid, J., and Ligon, J. M. (2000). The biosynthetic gene cluster for the
microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So
ce9O. Chemistry & Biology 7, 97-109.
Moore,R.E., Patterson,G.M.L., and Carmichael,W.W. (1988). New pharmaceuticals
from cultured blue-green algae. In Biomedical Importance of Marine Organisms,
D.G.Fautin, ed. (San Francisco: California Academy of Sciences), pp. 143-150.
Neilan, B. A., Dittmann, E., Rouhiainen, L., Bass, R. A., Schaub, V., Sivonen, K., and
Borner, T. (1999). Nonribosomal peptide synthesis and toxigenicity of cyanobacteria.
Journal of Bacteriology 181, 4089-4097.
Nogle, L. M. Structure elucidation and biosynthetic investigations of marine
cyanobacterial secondary metabolites. 2002. Oregon State University.
Ref Type: Thesis/Dissertation
Omura, S., Ikeda, H., Ishikawa, J., Hanamoto, A., Takahashi, C., Shinose, M.,
Takahashi, Y., Horikawa, H., Nakazawa, H., Osonoe, T., Kikuchi, H., Shiba, T.,
Sakaki, Y., and Hattori, M. (2001). Genome sequence of an industrial microorganism
Streptomyces avermitilis: deducing the ability of producing secondary metabolites.
Proceedings of the National Academy of Sciences of the United States of America 98,
12215-20.
On, P. T. and Jones, G. J. (1998). Relationship between microcystin production and
cell division rates in nitrogen limited Microcystis aeruginosa cultures. Limnology and
Oceanography 43, 1604-1614.
Patterson, G. M. L., Baker, K. B., Baldwin, C. L., Bolis, C. M., Caplan, F. R., Larsen,
L. K., Levine, I. A., Moore, R. E., Nelson, C. S., Tschappat, K. D., Tuang, G. D.,
Boyd, M. R., Cardellina, J. H., Collins, R. P., Gustafson, K. R., Snader, K. M.,
Weislow, 0. S., and Lewin, R. A. (1993). Antiviral activity of cultured blue-green
algae (cyanophyta). Journal of Phycology 29, 125-130.
Patterson, G. M. L., Baldwin, C. L., Bolis, C. M., Caplan, F. R., Karuso, H., Larsen, L.
K., Levine, I. A., Moore, R. E., Nelson, C. S., Tschappat, K. D., Tuang, G. D.,
Furasawa, B., Furasawa, S., Norton, T. R., and Rayboume, R. B. (1991).
Antineoplastic activity of cultured blue-green algae (cyanophyta). Journal of
Phycology 27, 530-536.
Patterson, G. M. L. and Bolis, C. M. (1995). Regulation of scytophycin accumulation
in cultures of Scytonema ocellatum. II. Nutrient requirements. Applied Microbiology
and Biotechnology 43, 692-700.
Patterson, G. M. L., Larsen, L. K., and Moore, R. E. (1994). Bioactive natural
products from blue-green algae. Journal of Applied Phycology 6, 15 1-157.
Pradella, S., Hans, A., Sproer, C., Reichenbach, H., Gerth, K., and Beyer, S. (2002).
Characterisation, genome size and genetic manipulation of the myxobacterium
Sorangium cellulosum So ce56. Archives of Microbiology 178, 484-92.
Quadri, L. E. N., Keating, T. A., Pate!, H. M., and Wa!sh, C. T. (1999). Assembly of
the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin: In vitro
reconstitution of aryl-4,2-bisthiazoline synthetase activity from PchD, PPchE, and
PchF. Biochemistry 38, 14941-14954.
Rapala, J., Sivonen, K., Lyra, C., and Niemela, S. I. (1997). Variation of microcystins,
cyanobacterial hepatotoxins, in Anabaena spp. as a function of growth stimuli.
Applied and Environmental Microbiology 63, 2206-22 12.
Repka, S., Koivula, M., Harjunpa, V., Rouhiainen, L., and Sivonen, K. (2004). Effects
of phosphate and light on growth of and bioactive peptide production by the
Cyanobacterium anabaena strain 90 and its anabaenopeptilide mutant. Applied and
Environmental Microbiology 70, 4551-4560.
Rossi, J. V., Roberts, M. A., Yoo, H.-D., and Gerwick, W. H. (1997). Pilot scale
culture of the marine cyanobacterium Lyngbya majuscula for its phannaceuticallyuseful natural metabolite curacin A. Journal of Applied Phycology 9, 195-204.
Rouhiainen, L., Paulin, L., Suomalainen, S., Hyytiainen, H., Buikema, W., Haselkorn,
R., and Sivonen, K. (2000). Genes encoding synthetases of cyclic depsipeptides,
anabaenopeptilides, in Anabaena strain 90. Molecular Microbiology 37, 156-67.
Salomon, C. E., Magarvey, N. A., and Sherman, D. H. (2004). Merging the potential
of microbial genetics with biological and chemical diversity: an even brighter future
for marine natural product drug discovery. Natural Product Reports 21, 105-121.
Sambrook,J., Fritsch,E.F., and Maniatis,T. (1989). Molecular Cloning: A Laboratory
Manual. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).
Sandmann, A., Sasse, F., and Muller, R. (2004). Identification and analysis of the core
biosynthetic machinery of tubulysin, a potent cytotoxin with potential anticancer
activity. Chemistry & Biology 11, 1071-1079.
Schneider, T. L., Shen, B., and Walsh, C. T. (2003). Oxidase domains in epothiolone
and bleomycin biosynthesis: thiazoline to thiazole oxidation during chain elongation.
Biochemistry 42, 9722-9730.
Schwartz, R. E., Hirsch, C. F., Sesin, D. F., Flor, J. E., Chartrain, M., Fromtling, R. E.,
Harris, G. H., Salvatore, M. J., Liesch, J. M., and Yudin, K. (1990). Pharmaceuticals
from cultured algae. Journal of Industrial Microbiology 5, 113-124.
Sharkey, F. H., Banat, I., and Marchant, R. (2004). Detection and quantification of
gene expression in environmental bacteriology. Applied and Environmental
Microbiology 70, 3795-3806.
Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T.,
Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H. (2002). Complete genome
sequence of Clostridium perfringens, an anaerobic flesh-eater. Proceedings of the
National Academy of Sciences of the United States of America 99, 996-1001.
Silakowski, B., Kunze, B., and Muller, R. (2001). Multiple hybrid polyketide
synthase/non-ribosomal peptide synthetase gene clusters in the myxobacterium
Stigmatella aurantiaca. Gene 275, 233-240.
Silakowski, B., Schairer, H. U., Ehret, H., Kunze, B., Weinig, S., Nordsiek, G.,
Brandt, P., Blocker, H., Hofle, G., Beyer, S., and Muller, R. (1999). New lessons for
combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene
cluster of Stigmatella aurantiaca DW4/3-1. Journal of Biological Chemistry 274,
37391-37399.
Silby, M. W. and Mahanty, H. K. (2000). The importance of recA mutant strains for
the study of antifungal genes in Pseudomonas aureofaciens PA147-2. Canadian
Journal of Microbiology 46, 744-748.
Song, J., Dong, F., Lilly, J. W., Stupar, R. M., and Jiang, J. (2001). Instability of
bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA
sequences. Genome 44, 463-469.
Stachelhaus, T., Mootz, H. D., and Marahiel, M. A. (1999). The specificity-conferring
code of adenylation domains in nonribosomal peptide synthetases. Chemistry &
Biology 6, 493-505.
Steege, D. A. (2000). Emerging features of mRNA decay in bacteria. RNA 6, 10791090.
Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids
Research 22, 4673-4680.
Tillett, D., Dittmann, B., Erhard, M., von Dohren, H., Borner, T., and Neilan, B. A.
(2000). Structural organization of microcystin biosynthesis in Microcystis aeruginosa
PCC7806: an integrated peptide-polyketide synthetase system. Chemistry & Biology
7, 753-764.
Tillett, D. and Neilan, B. A. (2000). Xanthogenate nucleic acid isolation from cultured
and environmental cyanobacteria. Journal of Phycology 36, 25 1-258.
Turgay, K. and Marahiel, M. A. (1994). A General Approach for Identifying and
Cloning Peptide Synthetase Genes. Peptide Research 7, 238-241.
Watanabe, M. F. and Oishi, S. (1985). Effects of environmental factors on toxicity of a
cyanobacterium (Microcystis aeruginosa) under culture conditions. Appl Environ
Microbiol 49, 1342-1344.
Williamson, R. T., Boulanger, A., Vulpanovici, A., Roberts, M. A., and Gerwick, W.
H. (2002). Structure and absolute stereochemistry of phormidolide, a new toxic
metabolite from marine cyanobacterium Phormidium sp. Journal of Organic
Chemistry 67, 7927-7936.
Zazopoulos, E., Huang, K., Staffa, A., Liu, W., Bachmann, B. 0., Nonaka, K., Ahiert,
J., Thorson, J. S., Shen, B., and Famet, C. M. (2003). A genomics-guided approach for
discovering and expressing cryptic metabolic pathways. Nature Biotechnology 21,
187-190.
CHAPTER III
CLONING AND BIOCHEMICAL CHARACTERIZATION OF THE
HECTOCHLORIN BIOSYNTHETIC GENE CLUSTER FROM THE MARINE
CYANOBACTERIUM LYNGBYA MAJUSCULA
INTRODUCTION
The marine envirorn-nent continues to offer a vast array of biologically active
natural products and constitutes a major source of molecules for drug discovery and
development (Newman et al., 2003). Among these organisms, marine cyanobacteria
have emerged as one of the richest producers of new natural product chemotypes with
potent biological properties (Burja et al., 2001; Gerwick et al., 2001). A predominant
metabolic theme in cyanobacterial metabolites is the rich integration of polyketide
synthases (PKS) and nonribosomal peptide synthetases (NRPS) (Gerwick et al., 2001).
PKS's and NRPS's represent a large group of multifunctional enzymes with a modular
organization that assemble complex molecules using acetate or amino acids as
building blocks, respectively. Additionally, these secondary metabolites exhibit
tremendous structural diversity due to the presence of numerous tailoring enzymes that
catalyze oxidation, reduction, methylation, different types of halogenation and
epimerization (Finking and Marahiel, 2004; Weissman, 2004). A complete
understanding of the biosynthesis of these compounds at the molecular genetic level
will provide key information for harnessing these gene clusters to produce novel
molecules through combinatorial genetic engineering.
Hectochlorin (54) was first isolated from a Jamaican isolate of the
cyanobacterium Lyngbya majuscula (designated as L. majuscula strain JHB). It was
subsequently re-isolated from L. majuscula collected at Bocas del Toro in Panama.
The planar structure of hectochiorin was elucidated by NMR methods and mass
100
spectral analyses, and its absolute stereochemistry was deduced by X-ray
crystallography (Marquez et al., 2002). Structurally, hectochlorin is related to
dolabellin (24) isolated from the sea-hare Dolabella auricularia (Sone et al., 1995)
and lyngbyabellin A (22) (Luesch et al., 2000b) and lyngbyabellin B (23) (Luesch et
al., 2000a; Milligan et al., 2000) from L. majuscula (Figure 111.1). Initial antimicrobial
assays indicated that hectochiorin was a potent antifungal agent. Its fungicidal activity
and potency against a number of plant pathogens initiated synthetic efforts and a total
synthesis of hectochiorin has been reported (Cetusic et al., 2002). Further testing
revealed that Ptk2 cells (derived from the rat kangaroo, Potorous tridactylus) treated
with hectochiorin showed an increase in the number of binucleated cells as a result of
arrest of cytokinesis, a result typical of compounds that cause hyperpolymerization of
actin. Hectochiorin was also evaluated against the in vitro panel of 60 different cancer
cell lines at the National Cancer Institute and showed potent activity towards cell lines
in the colon, melanoma, ovarian and renal sub-panels. It yielded a flat dose-response
curve against most cell lines, a characteristic of compounds that are antiproliferative
but not directly cytotoxic (Marquez et al., 2002). The unique biological properties and
presence of intriguing structural features in hectochiorin, such as the gem dichloro
group and two Dliv units, prompted us to clone and characterize its biosynthetic gene
cluster. In this study, we report the cloning, sequencing and partial biochemical
characterization of the hectochlorin biosynthetic pathway from
L.
majuscula ifiB.
101
S
CI
N
16J
iO
9
N
0
0
0
S
24
H7\25
OH
OH
o
20
Hectochlorin (54)
S
Dolabellin (24)
I
%O
HN
0
HNXO
N
HN
HN
H/TJH
Lyngbyabellin B (23)
Lyngbyabellin A (22)
OCH3
NO
R=BrJamaicamide A(27)
RHjamaicamide B (28)
HN)Lj
OCH3
"
NO
Jamaicamide C (29)
c)O
Figure 111.1: Chemical structure of hectochiorin (54) and related
metabolites, dolabellin (24), lyngbyabellin A (22) and lyngbyabellin B (23).
Jamaicamides A-C (27-29) are additional major metabolites isolated from L.
ma] uscula JHB.
102
RESULTS AND DISCUSSION
Construction of L. majuscula JHB genomic library
High molecular weight DNA for library construction was obtained as
described in Chapter II. The resulting high molecular weight DNA (40-45 kb)
(Figure 111.2) was used to generate a genomic DNA library using Epicentre' s
pCCFOS 1 fosmid cloning vector kit. The fosmid vector had a major advantage over
the cosmid in that it was maintained as a single copy within the cell and therefore was
more stable.
High
molecular
weight
40 kb T7
control DNA
High molecular weight
Genomic DNA from
L. majuscula JHB
40kb
Figure 111.2: High molecular weight genomic DNA from cultured L.
majuscula JHB. Genomic DNA was isolated by phenol extraction
followed by purification with 100/G Genome purification kit (Qiagen). The
resulting DNA (- 40 kb) was used for library construction.
103
PCR amplification of probes and screening of library
The structure of hectochiorin (54) suggested that it was derived from a mixed
PKS/NRPS pathway. C1-C8 were proposed to arise from intact acetate units
assembled by a PKS with the pendant methyl group (C9) added from S-adenosyl
methionine (SAM). Portions of the thiazole rings, C10-C12 and C18-C20 plus the
heteroatoms, were proposed to originate from cysteine (Chang et al., 2004; Sitachitta
et al., 1998). DHiv, an intermediate of valine biosynthesis, was proposed to form
Cl 3-C 17 and C2 1 -C25 units. Therefore, our initial strategy to isolate the hectochiorin
biosynthetic gene cluster involved the use of more general adenylation domain probes
based on A2 and A8 conserved motifs (Neilan et al., 1999). Unfortunately, this
strategy proved unsuccessful in isolating the hct gene cluster and instead led to the
identification of an approximately 20 kb cluster whose genetic architecture indicated
its involvement in the biosynthesis of an unrelated metabolite (A.V. Ramaswamy and
W.H. Gerwick, Chapter II).
Although chemical analysis of L. majuscula JHB has yielded the jamaicamides
A-C (27-29) (Edwards et al., 2004), hectochlorin (54) and 23-deoxyhectochlorin (K.L.
McPhail and W.H.Gerwick, unpublished data) as major metabolites, the presence of
multiple PKS/NRPS pathways was detected during library screening (D.J. Edwards
A.V. Ramaswamy and W. H. Gerwick, unpublished). Therefore, a more specific
approach was used, which focused on the modules involved in cysteine incorporation,
and specifically on the heterocyclization domains within these modules. Degenerate
primers spanning the heterocyclization domain and conserved region of the
adenylation domain (between core motifs A4 and A5) were designed based on
alignments of previously characterized heterocyclization domains (Figure III.3A).
These primers were used to amplify a 1.8 kb fragment which was cloned into pGEM-T
Easy vector (Figure III.3B). Several clones were sequenced and yielded three distinct
beterocyclization-adenylation domain products, thus indicating that there were three
different gene clusters or three distinct modules involved in the activation and
incorporation of cysteine in this L. majuscula genome.
104
90
BarG
CurF
Mt aC
FPITEQQAYWLGRNSHFDLGNITTHGYLELDCEN-LALDRLSQAWQQVIDHHDMLRMVI-FPLNDIQQAYWIGRNQI FDLGNIATHIYIEVDCEN-LNLESLHQAWRRLI DI-IHDMLRMVVFPLTDIQETYFVGRGTDFGI SGMSCHLYHELDTRG-MDVARLSRAWQRLIEQHVMLRSVVFPLTDIQNAYWVGRQGAFDAGGVAAJisYLELAFDT-LDLPRLES ILQRLIEYHDMLRMVV-FPL'rDIQGSYWLGRTGAFTvp- SGIHAYREYDCTD-LDVARLSRAFRKVVARHDMLRAHT-
MtaD
EpoB
Bim-Cys FPLTDVQRAYYVGREGGFALGGVSTHAYLEIEAPR-IDVARFTGALRGVIARHPMLRAVI-PchF
FPLTPVQAAYVLGRQAAFDYGGNACQLYAEYDWPADTDPARLEAAWIThNVERHPMLRAVI
HMWP2
FPLTPVQHAYLTGRMPGQTLGGVGCHLYQE FEGHC-LTASQLEQAITTLLQRHPMLHIAF-IletForward
BarG
CurF
MtaC
MtaD
EpoB
Bim-Cys
PchF
HMWP2
760
--ALNFDLSVYDI FGLLAAGGTLVMPTPEAAKDPVHWVELMTTHQVTLWNTVPALMQMLVEY
--ALNFDLSVYDIFGVLGAGGAIVMPPPKAPKDPACWRELI IAHEVTLWNSVPALMQMLVEH
--SLGFDLSVYDI FGSLAGAAIVLPQPEVTRDPAHWLQVLRQQGVTIWNSVPTLMEMLVDQ
-- SLS FDLSVYDLFGVLAAGGAIVMPEPGT SRDPGRWQVLLEKTGVT IWNSVPALMDMLVEF
--SLSFDLSVYDVFGILAAGGTIVVPDASKLRDPAHWAALIEREKVTVWNSVPALMRMLVEH
--SPSFDLAVYDLFGVLAAGGTVVvPAHDRRRDPGHWAELIRRERVTLWNSVPALGTLLTEY
--ELSFDLSVYDFFGATAAGAQVVLPDPARGSDPSHWAELLERHAITLWNSVPAQGQMLI DY
--ALHFDLSVYDI FGVLRAGGALVMVMENQRRDPHAWCELIQRHQVTLWNSVPALFDMLLTW
AdenReverse
Figure III.3A: Alignment of heterocyclization-adenylation domains for primer
design. Barbamide (BarG) and curacin A (CurF) from L. majuscula (Chang et
al., 2002; Chang Ct al., 2004), mxyothiazol (MtaC and MtaD) from Stigmatella
auriantica (Silakowski et al., 1999), epothilone (EpoB) from Sorangium
cellulosum (Molnar et al., 2000), bleomycin (Bim-cys) from Streptomyces
verticillus (Du Ct al., 2000b); pyochelin (PchF) from Pseudomonas aeruginosa
(Quadri et al., 1999) and high molecular weight protein 2 (HMWP2) from
Yersinia pestis (Gehring et al., 1998). Conserved residues used for primer
design are highlighted. Primers were designed using the CODEHOP strategy
(Rose et al., 1998).
1.8kb +
Figure III.3B: PCR amplification of heterocyclization domain probe with
Hetforward and Adenreverse resulted in the amplification of a 1.8 kb fragment.
105
An equal concentration of these three DIG-labeled probes was used to screen a
library of approximately 2800 clones, which identified 12 different fosmids at the
colony hybridization level. Restriction digest mapping with Hindlil and EcoRI
enzymes and Southern hybridization analyses using the heterocyclization-adenylation
domain probe led to the identification of two overlapping fosmids, pHctl and pHctII as
candidates for the hct gene cluster. To determine if these fosmids contained PKS
elements, a pair of degenerate primers based on the -ketosynthase (KS) domain were
used to amplify a 0.7 kb fragment from this domain (Beyer et al., 1999). Sequence
analysis of the PCR products indicated that the two fosmids contained the same KS
domain. These results were consistent with our findings that the starter unit in the
jamaicamide A (5) pathway was a hexynoic acid that becomes extended by a single
acetate derived extension (Edwards et al., 2004). Therefore, we predicted that the
starter unit in hectochiorin biosynthesis might be a hexanoic acid that undergoes a
single ketide extension. Additionally, end-sequencing of these two fosmids revealed
that 3'-end of fosmid pHctl encoded a heterocyclization domain and 5'-end of fosmid
pHctII contained a putative halo genase related to BarB I from barbamide gene cluster
(Chang et al., 2002). Because, this genetic architecture was consistent with our
predictions for hectochiorin biosynthesis, fosmids pHctl and pHctII were selected for
further sequence analysis.
106
DNA sequencing and analysis of the hectochiorin biosynthetic gene cluster
The DNA sequences of the two overlapping fosmids, pHctl and pHctII, were
determined by shotgun cloning and assembled into a 42 kb contiguous region, which
contained the 38 kb hct gene cluster (Figure 111.4). Flanking the hct cluster at the 5'-
end are three small ORFs (I, II and III). ORF I encodes a 238 amino acid reverse
transcriptase, which is 58% identical to a similar protein from Trichodesmium
erythraeum, ORF II encodes a putative endonuclease with 50% identity to a similar
protein from Anabaena variabilis and ORF III is similar to a hypothetical protein from
Nostocpunctiforme PCC73102. At the 3'-end are two small ORF's (IV, V) that also
encode hypothetical proteins from Trichodesmium erythraeum and helped to confirm
the outer boundaries of hectochlorin biosynthetic gene cluster. The gene cluster
exhibits a relatively low overall G+C content (-S 42%), which is comparable to the
G+C content encountered in cyanobacterial genomes (http://www.kazusa.or.jp/cyano)
and other secondary metabolite gene clusters from L. majuscula (Chang et al., 2002;
Chang et al., 2004; Edwards and Gerwick, 2004; Edwards et al., 2004).
ORFs hcf
11111 A B C
D
F
E
G H Iv
v
pHctl
DHCtII
3 kb
42kb
Figure 111.4: Genetic map of the hct gene cluster spanning 38 kb. Inserts from
two overlapping fosmids, pHctl and pHctII were sequenced to map the hct cluster.
107
Table III.!: Deduced functions of the proteins in the hct biosynthetic gene cluster
Protein
Proposed function
ORF I
Size (amio
acids)
-238
ORF II
119
HNH endonuclease
ORF III
51
hypothetical protein
HctA
606
acyl-ACP synthetase'
reverse transcriptase
Sequence similarity
(protein, organism)
Trichodesmium
erythaeum
Anabaena variabilis
ATCC 29413
Nostocpunct?forme
ATCC 73102
JarnA
Identity!
Similarity
58%, 73%
50%, 65%
60%, 73%
60%, 78%
L. majuscula JHB
Hct B
484
putative halogenase,
acyl carrier protein
HctC
408
transposase
HctD
1924
polyketide synthase
HctE
3356
nonribosomal peptide
synthetase
nonribosomal peptide
synthetase
cytochrome P450
monooxygenase
BarBi
29%, 47%
L. majuscula 19L
JamC
L. mafuscula JHB
35%, 54%
TnpB
Helicobacterpylori
JamL
33%, 55%
HctF
3945
HctG
447
I-IctH
444
cytochrome P450
monooxygenase
L.majuscula 19L
BarEfBarG
L.majuscula 19L
TaH (P450
hydroxylase)
Edwards et
al., 2004
Chang et al.,
2002
Edwards et
at., 2004
Cesini et al.,
1996
56%, 72%
L.majuscula JHB
BarE/BarG
Reference
46%, 63%
57%, 71%
45%, 62%
60%, 72%
36%, 54%
Myxococcus xanthus;
(P450 monooxygenase)
Polyangium cellulosum
35%, 56%
(P450 monooxygenase)
Polyangium cellulosum
36%, 57%
TaH (P450
hydroxylase
Myxococcux xanthus
34%,
Edwards et
al., 2004
Chang et at.,
2002
Chang et al.,
2002
Paitan et al.,
1996
spirangiene
biosynthesis,
Accession no.
AJ505006
spirangiene
biosynthesis,
Accession no.
AJ505006
Paitan et al.,
1996
ORF IV
90
hypothetical protein
Trichodesmium
erythraeum
70%, 83%
ORF V
222
hypothetical protein
Trichodesmium
eiythraeum
58%,75%
108
The
hct
gene cluster consists of eight ORFs with the first ORF
hctA
encoding a
protein that is similar to acyl-ACP synthetases (Table 111.1). Sequence analysis
revealed a 59% identity to JamA in the jamaicamide biosynthetic gene cluster
(Edwards et al., 2004). Next, the product of hctB has two distinct domains. The Nterminal domain shows closest similarity to barbamide BarB 1 (29% identity), (Chang
et al., 2002) coronamic acid CmaB (33% identity) (Ullrich and Bender, 1994) and
syringomycin SyrB2 (33% identity) (Guenzi et al., 1998; Zhang et al., 1995). It also
shows weak homology to Fe2/ 2-oxoglutarate-dependent hydroxylases like phytanoyl
Co-A hydroxylases (PAHX) and possesses all of the conserved amino acids necessary
for co-factor binding (Figure 111.5) (Mukherji et al., 2001). The C-terminal domain is
35% identical to JamC, an acyl carrier protein in the j amaicamide gene cluster
(Edwards et al., 2004).
121
BarBl
PAUX
BarB2
SyrB2
CmaB
HctB
.
.
.
.
.
.
150
. SYDR HLDIQALSDI ISHPKIAHRV
. MITKVQD FQEDKELFRY CTLPEILKYV
. NKMNYDR RLDIKALNDI ISHPKVVDRV
GT.NIAUYDR HLDDDFLASH ICRPEICDRV
. ITNYDR ULDVDLLTSH VFRXEIVHRV
VLNU4TSRDL HLFHQPIAEM FKNNKIVRVL
.
.
.
171
200
BarBi GDEG. TDWHQ AESFVEFEGK SK. . . LVPTE
PAHX DSGXXTSRHP LHQDLHYFPF RP ..... S..
BarB2 GDEG. TDWHQ AUSFVEFEGE SK. . . LVPTE
SyrB2 GDEG. TDWHQ ADTFISGX PQ. . . IIWPE
CmaB GNEG.TDWHQ ADTFAHAPAS RN. . .WCAR.
HctB GEGE. IKWHQ VYIDSYDPSAY DPQKPALLYP
301
330
BarBl FTSRGSE PNTSGSS ER YGWSTRPVPT
PAHX
BarB2
SyrB2
CmaB
HctB
FHPLLIHGS
GQNKTQGFR KAISCHFASA
FTSRCMHGSE PNTSESS . IR YGWSTRPVST
FWSTLMHASY PHSGESQEMR MGFASRYVPS
FGSMLZ.PSSL PNSTTDK ....... TAWP.4S
FTERVMHSSP PNKSKQQ ER LGINGRYVPP
Figure 111.5: Alignment of barbamide BarBi and B2, syringomycin
SyrB2, coronamic acid CmaB and PAHX with HctB. All enzymes
share putative conserved residues (highlighted) involved in binding
iron (II) and 2-oxoglutarate cofactors.
109
The hctC gene is similar to insertion sequence (IS) elements from other
bacteria and encodes for a putative transposase. BLAST analysis of the transposase
showed that it was most similar
(P3
1% identity) to TnpB encoded by an 1S605 type
element from Helicobacter pylon (Censini et al., 1996), and transposases encoded by
other IS elements such as IS 1341 from the thermophile PS3 (Murai et al., 1995),
1S891 from the cyanobacterium Anabaena M- 131 (Bancroft and Wolk, 1989), and
1S1136, from the erythromycin gene cluster in Saccharopolyspora erythraea (Donadio
and Stayer, 1993).
About 50 bp downstream from hctC is located hctD, which encodes a type I
PKS module with KS-AT-CM-KR-ACP domain organization. The hctD gene shows
highest similarity to PKS genes from the jamaicamide A and curacin A biosynthetic
pathways (Chang et al., 2004; Edwards et al., 2004) and all five domains show typical
conserved core motifs when aligned with PKS genes from these two gene clusters
(Figure 111.6).
A
B
KS-HctD LHGPSLAIDTACSSSL- --YVEAEGT---IGBLEAAA
KS-JamJ LRGPVMTVETASA3L- - -YVEAHGT-- -IGHASTAA
AT-HctD YTQPALFALEYALCQL- - - IGHSVGE
AT-Jainj YTQPVLFAIEYALCQL-- -MGHSVGE
AT-JamK YTQPCLFALEYALCQL- - -MGHSVGE
AT-CurF YTQPALFAIEYALFKL- - -MGHSVGE
AT-CurG YTQPALFATEThLFKL- - -MGHSVGE
Cons.
CHSVC
KS-JamK LIGPNLAVDTACSSSL- - -YLF.AfiGT- - -IGH'rEGAA
KS-CurF LKGPVVAVEAACSSSL- -YLEAHGT- - -IGNAATAA
KS-CurG LTGPAITVQTACSTSL- -YV.?.BGT- - -VGHLDAAA
(malonyl CoA)
C
C-MT-HctD LLEIGAGGGTT
D
YLITGGLGAL- -
C-MT-JamJ LLEIGGTGTT
C-MT-CurJ LLEIGAGTGGTT
KR-CurH
KR-JamJ
KR-CurG
KR-HctD
KR-Hct
KR-HctF
KR-Ces
KR-JamP
YLITGGLGYL- -- -AVD---
YLITGGTGFL- - -ASD-
YLISGGLGGI----DVD--YLISGGLGGI----AVD--YVVTGGLGGI----DGD--YLITGGLGGI----CAD---
Figure 111.6: Sequence alignments of PKS domains from HctD with other
cyanobacterial PKS's (jamaicamide A (5) and curacin A PKS genes). KS domain
alignment (A), AT-domain specific for malonyl-CoA (B), alignment of C-methyl
transferase (C), alignment of KR domains from jamaicamide A, curacin A,
cereulide and hectochlorin biosynthetic gene clusters (D).
110
The stop site of hctD overlaps with the start site of the next ORF
hctE,
which
encodes for a bimodular NRPS. Initial analysis of the first module revealed the
presence of a KR domain embedded between the A and PCP domains, which is
unprecedented in NRPS modules. The A-domain is most similar to the A-domain
from BarE of the barbamide biosynthetic pathway (46% identity) (Chang et al., 2002)
and also shows similarity to A-domains from the microcystin McyG and nodularin
NdaC clusters (41% identity) (Tillett et al., 2000; Moffitt and Neilan, 2004). A
notable feature of the residues that contribute to substrate specificity in the A-domain
is substitution of the conserved aspartate residue (Asp23 5) involved in interactions
with the amino group of the cognate amino acid by valine (Va1235) at this position.
BLAST analysis of the KR domain revealed it had weak similarity to KR domains
from PKS (23% identity), but higher similarity to a KR from the partially
characterized cereulide biosynthetic gene cluster (31% identity) (Ehling-Schulz et al.,
2005). The second module in HctE contained all of the required domains for the
adenylation and heterocyclization of cysteine and is most similar to BarG from the
barbamide biosynthetic gene cluster (57% identity) (Chang et al., 2002) and CurF
from the curacin A biosynthetic pathway (58% identity) (Chang et al., 2004).
This module also contains an FMN-dependent oxidase that is embedded between the
A9 and AlO conserved motifs of the adenylation domain, which is likely involved in
the fonnation of thiazole ring (Du et al., 2000a).
111
Next in the cluster is hctF, a bimodular NRPS whose domain architecture is
identical to that of HctE. The predicted HctE and HctF proteins share a 76% overall
identity. The hctE and hctF genes also share overlapping start and stop sites,
suggesting that hctDEF are transcribed as an operon. A thioesterase domain is located
at the C-terminal end of HctF and is proposed to catalyze hydrolysis from the
biosynthetic assembly line and cyclization.
The next ORF, hctG, begins about 600 bp downstream from the end of hctF
and encodes for a putative P450 monooxygenase. The hctG gene is followed by
another ORF, hctH, whose product also shows similarity to putative P450
monooxygenases. While HctG and HctH are most similar to each other (47% identity)
they also show similarity to a P450 monooxygenase involved in spirangiene
biosynthesis (unpublished, accession number: AJ505006) (HctG 35% identity, HctH
36% identity) and TaH, a P450 hydroxylase from the TA biosynthetic gene cluster
(HctG 36% identity, HctH 34% identity) (Paitan et al., 1999a).
112
Cloning, expression and functional analysis of adenylation domains from the
hectochiorin biosynthetic gene cluster
In order to obtain biochemical confirmation of the predicted adenylation
domain specificities, both A-domains from HctE were overexpressed as 6x His-fusion
constructs, purified and analyzed using the ATP-PPi exchange assay (Stachelhaus and
Marahiel, 1995). Constructs for protein expression were based on the start and stop
sites of BarE and BarG constructs which have been expressed successfully (Chang et
al., 2002). The adenylation domains were PCR amplified and cloned into the
pET2Ob(+) expression vector. When E. coli BL2 1 (DE3) cells harboring these
pET2Ob(+) constructs were grown at temperatures above 20°C, only insoluble protein
was obtained. However, soluble protein could be generated when cells were grown at
18°C for 12-18 hours without IPTG induction (Figure III.7A). These A-domains were
designated as HCtEOIVA and HctE, respectively. Western blot analysis using a-His
antibodies was performed to confirm heterologous protein expression (Figure III.7B).
B
HctEQIVA
40
Figure 111.7: Overexpression and purification of adenylation domains in HctE
and Western blot analysis. HctEAOIVA (66.9 kDa) and HctEAc (56 kDa)
were overexpressed as 6x His-fusion proteins and purified (A). Western blot
analysis using a-His antibodies (B).
113
The relative activities of the various amino acid substrates in the ATP-PPi
exchange assay are displayed in Figures III.8A and III.8B. This assay is based on the
reversible nature of the adenylation reaction (Figure 1.2, Chapter I). The recognition
of a cognate amino acid by the adenylation domain results in the formation of
corresponding acyladenylate and inorganic pyrophosphate (Figure 1.2). The reaction
is reversible and the acyladenylate can undergo conversion to form the amino acid and
ATP. The use of radioactive pyrophosphate results in its incorporation into the
resulting ATP (AT32P) which is bound to charcoal and radioactivity is a measure of
the ability of an adenylation domain to activate a particular amino acid.
The first adenylation domain in HctE was found to activate 2-oxo isovaleric
(0-NA) acid to the greatest extent; however, it also activated 2-hydroxy isovaleric
acid (2-NA), 2-oxo-3-hydroxy isovaleric acid and DHiv to an appreciable level, thus
indicating that it possesses fairly relaxed substrate specificity (Figure III.8A). Several
other standard amino acid substrates were activated to a much less degree. These
results are consistent with the arrangement of amino acid residues in the binding
pocket of the A-domain in which the conserved Asp235 involved in interaction with
the amino group of the amino acid substrate is replaced by Va1235, a substitution
typical for a keto-acid or hydroxy-acid substrate (Stacheihaus et al., 1999). Because
2-OH WA and 2,3-DHiv were racemic mixtures in which each enantiomer was
represented at half concentration, the results of these experiments presented above are
not an accurate representation of adenylation domain specificity. Experiments using
R- and S-enantiomers of 2-OH NA and 2,3 DHiv are currently underway in order to
further determine the specificity of the adenylation towards these enantiomers.
In the case of the second adenylation domain, L-cysteine was activated to the
highest level (100%) and L-serine was activated up to 40%, while activation of Lglycine, L-tryptophan, L-histidine were equivalent to background (Figure III.8B).
114
a
HO'f
A
120
0
r
100
80
60
4o
20
0
2-oxo
IVA
2-oxo-3OH IVA
2-OH
IVA
2,3 diOH IVA
Val
2-oxo
Leu
Leu
Ala
His
Trp
Gin
B
120
1
100
- 80
>
60
>
0
40
20
_________________
0-4
Cys
Ser
Giy
Trp
His
Figure 111.8: Results of the ATP-PPi exhange assay for HCtEAO.JVA (A) and
HctEAc (B) and represent average of duplicate experiments.
Abbreviations: 0-IVA, 2-oxo-isovaleric acid; 2-OH IVA, 2-hydroxy-isovaleric
acid; 2-oxo-3-OH IVA, 2-oxo-3-hydroxy isovaleric acid; 2,3-diOH IVA, 2,3dihydroxy isovaleric acid; Va!, L-valine; 2-oxo-Leu, 2-oxo L-leucine; Leu, Lleucine; Ala, L-alanine; His, L-histidine; Gln, L-glutamine; Trp, L-tryptophan; Cys,
L-cysteine; Ser, L-serine; Gly, L-glycine.
115
Cloning and overexpression of HctA (acyl-ACP synthetase)
In order to examine the substrate specificity of HctA, the entire ORF was
cloned and expressed as 6x His-fusion protein in E. coli. Optimal protein expression
was observed at 25°C and induction with 0.4 mM IPTG (Figure 111.9). The protein
was purified to near homogeneity using Ni2taffinity chromatography and awaits
further characterization through ATP-PPi exchange assays. Methods to synthesize
5,5-dichiorohexanoic acid, an important substrate for this assay are currently being
explored at Christine Willis's laboratory at the University of Bristol, UK.
kDa
1
2
3
4
5
-
6
7
68.4 kDa
50
20
Figure 111.9: Overexpression and purification of HctA. Protein marker (1),
total protein extract (2), insoluble fraction (3), soluble fraction (4), eluted
HctA (5,6) and dialyzed HctA (7).
HctA
HctE
HctD
Module I
HctB
Module 2
HctF
Module 3
K
H
APC
AMP
S
HO)
OH
HO)(
Cl
CI
OH
Enzymatic domains:
C
Condensation
AS AcyI-ACP synthetase A
Adenylation
Hal Putative halogenase
PCP Peptidyl carrier protein
ACP Acyl carrier protein
Cy Cyclization
KS Ketosyrithase
AT Acyltransferase
CM C-methyl transferase
KR Ketoreducatse
Q Module 2
Ox
TE
CI
Oxidase
Thioesterase
modification?
CI
,-
HctG or HctH
'o
Post-N RPS
Cl
Oxy P5O monooxygenase
A
Module 4
HctGorHctH
02
0
>-
>t?O
OH
HO).
OH
(Y
W PI'ø..- W
>O
"T'
>-:
d120
HO)
Figure 111.10: The proposed biosynthesis of hectochiorin. Insets 1 and 2: In modules 2 and 4, the adenylation domains
are proposed to activate 2-oxo-isovaleric acid, which is reduced in situ to 2-hydroxy isovaleric acid by the embedded
KR domain and further oxidized by HctG or HctH to form 2,3-dihydroxy isovaleric acid.
117
Proposed biosynthesis of hectochiorin
This report describes the cloning and preliminary characterization of 38 kb of
DNA from the marine cyanobacterium Lyngbya majuscula that is putatively involved
in the biosynthesis of hectochiorin (1). The overall genetic architecture and domain
organization encountered in this cluster is consistent with the arrangement expected
for the biosynthesis of hectochiorin, and its identity is further strengthened by
biochemical characterization of the adenylation domains. The presence of a hybrid
histidine kinase/response regulator protein about 10 kb upstream of the hct gene
cluster leads us to speculate that the expression of this cluster is probably regulated by
signal transduction mechanism, although there is no evidence to support this. Indeed
to date, there is very little understanding of the regulation of natural product
biosynthetic pathways in cyanobacteria.
HctA, an acyl-ACP synthetase homolog, is proposed to activate free hexanoic
acid in an ATP-dependent maimer and thus initiate hectochiorin biosynthesis (Figure
111.10). HctA shares a high similarity to j amaicamide JamA (obtained from the same
L. majuscula strain), which has been shown to biochemically activate 5-hexynoic acid
through an ATP-PPi exchange assay (Edwards et al., 2004). The acyl group is then
likely transferred from the acyl-adenylate to the ACP domain in the C-terminal region
of HctB. The N-terminal region of HctB shows similarity to barbamide BarB 1 and B2
(Chang et al., 2002), coronamic acid CmaB (Ullrich and Bender, 1994), and
syringomycin SyrB2 (Zhang et al., 1995) and weak similarity to phytanoyl-CoA
hydroxylases (Mukherji et al., 2001). Sequence alignments of these proteins with
HctB show that they share conserved residues involved in co-factor binding,
suggesting that the enzyme mechanism may require iron (II) and 2-oxoglutarate as cofactors (Figure 111.5) (Mukherji et al., 2001; Kershaw et al., 2001). BarBiand B2 are
believed to mediate the transformation of leucine to trichioro-leucine during the
biosynthesis of barbamide (Chang et al., 2002). Similarly, SyrB2 is postulated to be
involved in the chlorination of threonine during syringomycin biosynthesis (Zhang et
al., 1995) and CmaB in the conversion of isoleucine to coronamic acid in coronatine
biosynthesis (Ulirich and Bender, 1994). Hence, we speculate that HctB is involved in
118
the formation of the gem dichioro group in hectochiorin based on its location in the
cluster and proposed biochemical properties.
Located between hctB and hctD is an IS element that encodes for a putative
transposase. IS elements are fairly small segments (1.5 -2.0 kb) with a simple genetic
organization which encode functions involved in their mobility (Mahillon and
Chandler, 1998). IS elements are believed to play a major role in the plasticity of
bacterial genomes and are often involved in the assembly of gene clusters with
specialized functions such as pathogenicity, antibacterial activity or catabolic
pathways (Mahillon et al., 1999). The occurrence of transposases at the boundaries of
several cyanobacterial gene clusters has been previously reported (Christiansen et al.,
2003; Edwards et al., 2004; Moffitt and Neilan, 2004; Tillett et al., 2000; Becker et al.,
2004). However, it is interesting to note that the HctC transposase is present within the
putative hct gene cluster. It is likely that these transposases may play an important
role in the acquisition and distribution of secondary metabolite gene clusters between
different strains and genera.
The chlorinated hexanoate initiator of hectochiorin biosynthesis is next
proposed to undergo one round of ketide extension by the PKS module (KS-AT-CM-
KR-ACP) found in HctD. The AT- domain in this module appears to be specific for
malony!-CoA (Figure III.6B) (Haydock et al., 1995). The C-MT transferase possesses
a signature motif (LExGxGxG) that is also present in the C-methyl transferases from
the jamaicamide A and curacin A biosynthetic gene clusters (Figure III.6C). This is
consistent with precursor feeding experiments to several polyketide pathways in which
methyl branching at C-2 positions of several cyanobacterial polyketides all derive
from SAM (Gerwick et al., 2001; Chang et al., 2004; Edwards et al., 2004).
119
Table 111.2: Substrate specificity of A domains from the first modules in HctE
and HctF. The binding pocket amino acids involved in substrate recognition
determined using the method of (Stacheihaus et al. 1999 and Challis et a!, 2000) of
A-domains from the first module of HctE and HctF (this study) were compared to
residues from previously characterized A-domains BarE (Chang et al., 2002)
McyG (Tillett et al., 2000)and NdaC (Moffitt and Neilan, 2004) involved in
A-
Binding pocket amino acids
domain
Substrate
235
236
239
278
299
301
322
330
V
G
V
W
L
A
L
F
V
G
V
W
L
A
L
F
BarE-A
V
G
I
I
V
G
G
T
NdaC-A
V
G
W
V
A
A
S
McyG-A
V
G
I
I
2-oxoisovaleric acid
2-oxoisovaleric acid
2-oxo-leucic
acid
phenylacetate
W
V
A
A
S
phenylacetate
GrsA-Phe
D
A
W
T
I
A
A
I
phenylalanine
HctE-A0.
IVA
HctF-A0.
IVA
Next, HctE is composed of two NRPS modules that are proposed to
incorporate DHiv and cysteine. Module 2, which presumably catalyzes the
incorporation of DHiv, is unique in that it possesses a KR domain embedded between
the A and PCP domains. Analysis of the A-domain binding pocket residues which
confer substrate specificity using the previously described methods (Challis et al.,
2000; Stacheihaus et al., 1999) revealed that the conserved Asp235 involved in ionic
interaction with the amino group of the substrate amino acid was replaced by Va1235
(Table 111.2). This type of codon specificity suggests a preference for a non a-amino
acid substrate such as a hydroxy- or keto-acid. Furthermore, this A-domain is most
closely related to the barbamide BarE A-domain, which also lacks the conserved
Asp235 and shows high specificity for both the non-chlorinated and trichiorinated 2oxo derivatives of L-leucine (P.Flatt and W.H.Gerwick, unpublished) and only modest
activation of corresponding a-amino acids (Chang et al., 2002). Other previously
characterized cyanobacteria! A-domains with the missing Asp235 residue include
McyG and NdaC A-domain, both of which are involved in activating a phenylacetate
120
starter unit in the biosynthesis of microcystin and nodularin, respectively (Table 111.2)
(Moffitt and Neilan, 2004; Tillett et al., 2000). Biochemical support for HCtEAOWA
function was obtained through the ATP-PPi exchange assay in which the A-domain
was found to activate the 2-oxo derivative of valine over other substrates (Figure
III.8A). The exchange assays also showed that this A-domain activates 2-OH WA, 2oxo-3-hydroxy isovalerate and DHiv, suggesting that the A-domain has a fairly
relaxed substrate specificity.
Based on its location within module 2 in HctE, we propose that the embedded
KR domain may be involved in the in situ reduction of 2-0 WA to 2-OH WA (Figure
111.10 Inset 1). Recently, the presence of a KR domain in an NRPS module has been
reported in the partially characterized cereulide gene cluster (ces) from Bacillus
cereus
and is believed to be involved in the formation of 2-WA (Ehling-Schulz et al., 2005).
In cereulide biosynthesis, this has been validated by labeled precursor feeding studies
which concluded that 2-OH WA acid is derived from L-valine and is reduced to 2-
WA during the biosynthetic process (Kuse et al., 2000). Although the HctE and Ces
KR exhibit only weak similarity to KR domains from PKS modules, they do possess
all the key conserved amino acid residues required for enzymatic catalysis (Figure
III.6D) (Reid et al., 2003).
Next, 2-oxo-IVA undergoes 3-hydroxylation, a reaction presumably catalyzed
by either HctG or HctH. HctG and HctH are heme-dependent cytochrome P450
monooxygenases with conserved C-terminal motifs, GxHxCxGxxLxR, which are
involved in heme-binding and show greatest similarity to TaH from the TA antibiotic
biosynthetic gene cluster (Varon et al., 1992) and a P450 monooxygenase from
spirangiene biosynthesis (unpublished, accession #: AJ505006). TaH is responsible
for a specific post PKS hydroxylation at C-20 of antibiotic TA (Paitan et al., 1999b;
Paitan et al., 1999a). P450 monooxygenases involved in post assembly modification
are also seen in other polyketide gene clusters such as those involved in the
biosynthesis of erythromycin (Andersen and Hutchinson, 1992) and epothiolone
(Molnar et al., 2000). However, P450 monooxygenases that function during
preassembly to generate -hydroxyl amino acid substrates are encountered in NRPS
121
gene clusters such as those involved in the biosynthesis of the coumarin antibiotics
(Steffensky et al., 2000; Wang et al., 2000). A common theme in these latter gene
clusters is the occurrence of a heme oxygenase in conjunction with an A-PCP
didomain. The substrate amino acid is activated by the A-domain and is transferred to
the holo-PCP domain to yield an aminoacyl-S-enzyme intermediate which is f3hydroxylated by the heme monooxygenase. In novobiocin biosynthesis, NovH (APCP) activates the amino acid tyrosine to L-tyrosyl-AMP, which accumulates on its
PCP domain. Its partner enzyme, NovI, is a heme dependent cytochrome P450
monooxygenase that specificially hydroxylates this intermediate to form f3-
hydroxytyrosine (Chen and Walsh, 2001). Similarly, during biosynthesis of the
nucleoside antibiotic nikkomycin, NikPl activates histidine and the L-histidinyl-SNikP 1 serves as a substrate for the heme monooxygenase, NikQ, which introduces the
3-hydroxyl group (Chen et al., 2002). Such heme monooxygenases are thought to
have evolved in conjunction with corresponding NIRPS's to act on substrates that are
presented as L-aminoacyl-S-enzyme complexes, thus ensuring that only a select pool
of the substrate is modified (Chen et al., 2001). Although a A-PCP didomain scaffold
is not adjacent to the heme oxygenase in this cluster, it is plausible that HctG and/or
HctH may catalyze the 3-hydroxylation of 0-NA
in trans
during the preassembly
process (Figure 111.10 Inset 1). It is interesting to note that there are two heme
dependent oxygenases in the hct gene cluster and the extent of their involvement,
timing of 3-hydroxylation and biochemical mechanism await further characterization.
122
The second module in HctE shows high similarity to NRPS's specific for the
incorporation and heterocyclization of cysteine. Analyses of the amino acids in the
binding pocket of the adenylation domain are consistent with conserved amino acid
residues for cysteine recognition (Table 111.3). Notably, the condensation domain in
this module shows the presence of conserved core motifs required for
heterocyclization (Figure 111.11) (Schwarzer et al., 2003). In vitro biochemical
characterization of the HctEAc domain demonstrated that it activates L-cysteine
preferentially (Figure III.8B). An FMN-dependent oxidase domain is embedded
between the A9-A10 core motifs of the adenylation domain and presumably catalyzes
the oxidation of the thiazoline to a thiazole. Similar oxidase domains involved in
thiazole ring formation from the epothilone and bleomycin gene clusters have been
biochemically characterized and show high similarity to this domain from the hct gene
cluster (48% identity) (Schneider et al., 2003).
Table 111.3: Substrate specificity of the adenylation domains in the second
modules present in HctE and HctF. Amino acid residues involved in substrate
recognition from adenylation domains of the second modules of HctE and HctF
were identified using the method of Stachelhaus et al. 1999 and Challis et a!,
2000 and compared to other cyanobacterial A-domains involved in activating
cysteine BarGA2 and CurF. The numbering of binding pocket amino acid
residues correspond to GrsA-Phe A-domain numbering.
A-domain
Binding pocket amino acids
Predicted
substrate
235
236
239
278
299
301
322
330
HctE-Ac
D
L
Y
N
L
S
L
I
cysteine
HctF-Ac,
D
L
Y
N
A
L
L
I
cysteine
BarG-Ac
D
L
Y
N
L
S
L
I
cysteine
CurF-Ac,
D
L
Y
N
V
D
L
I
cysteine
123
Zi
Z2
C3
Z3
HetEcyc FPLTDIQQAYWLGR---RAVVLPDGQQ---flALLADSWS---LPPAPELP--HctFcyc FPLTDIQQAYWLGR- - -RAVVLPDGQQ- - -DALThDGWS- - -LPPAPELP- -
BarGcyc FPLTEIQQAYWLGR---RMVILPNGEQ---DALIP.DAWS---LPPAPELP--CurFcyc FPLNDIQQAYWIGR- - -RI4VVLADGNQ- - -DMLXFDAWS- - -LPPAPEIP - -
Cons.
FPLTxxQxAYxxGR- - -RAx.xxGxQ-
- -DxxxxDxxS- - -LPxxPxxP- - -
HctEcyc TNSTVLLWADILTCW- - -GDFT- - -GVVFTSVL- - -QVWLDHQVLEEQGELFQWD
HctFcyc TNSTVLLAADVLTP.W- - -GDT-- -GFTSTL---QVWLDHQVEQGLFQWD
BarGcyc TPSGLLAMiDFIAYW- - -GDFT- - -GVVFTSTL- - -QVWLDNSVAEQNGALLLIW
CurFcyc TPSGVgSAFASVLNYW- - -GT- - -GVSTL- - -QVLLDHrEEKGALAPSW
Cons.
TPXXXLXXXXXXVLXXW- - -GDFT- - -GVVFTSxL- - -QVxLDxQxicxxxxxxxxxW
Figure 111.11: Alignment of heterocyclization domains from barbamide BarG
and curacin A CurF with heterocyclization domains from the hct gene cluster
show presence of consensus core motifs.
Next, modules 4 and 5 in HctF are believed to catalyze the incorporation of the
second DHiv and cysteine units, respectively. The modular organization of HctF is
identical to that present in HctE. The A-domain in module 4 exhibits a remarkably
high similarity to the A-domain in module 2 (95% identical) with both adenylation
domains sharing identical binding pocket amino acid residues (Table 111.2). Thus, the
A-domain in module 4 is expected to catalyze the activation and incorporation of the
second 0-IVA that is reduced in situ to 2-OH IVA by the embedded KR domain and
then further oxidized by HctG or HctH to form DHiv (Figure 111.10 Inset 2). Although
the incorporation of the two DHiv units is catalyzed by modules that have the same
domain organizations, it is intriguing that their regiochemical orientation is different,
with the secondary alcohol of DHiv-1 forming the ester with the preceding polyketide
unit and the tertiary alcohol of DHiv-2 forming the ester with the preceding cysteine-
derived residue. It is unknown how the alternate orientations of DHiv are achieved by
these very similar modules.
Module 5 in HctF appears to catalyze the incorporation of a second L-cysteine
unit into hectochlorin (1). The A-domain binding pocket amino acids are in consensus
124
for the recognition of cysteine (Table 111.3) (Challis et aL, 2000). Conserved regions
were again identified for the heterocyclization domain, which catalyzes the cyclization
of cysteine (Figure 111.10). An oxidase domain is present between the A9 and Al 0
conserved motifs and is again likely involved in the oxidation of thiazoline to thiazole.
Finally, an embedded type I thioesterase which possesses conserved GxSxG motif is
present at the C-terminal end of HctF, and may be involved in catalyzing the release
and cyclization of hectochiorin. A candidate gene product to introduce the acetate
group at C-14 in hectochlorin (1) is lacking and leads us to speculate that this may be
catalyzed by an acetyl transferase not present in the hct gene cluster and likely as a
post-NRPS modification.
The natural products chemistry of marine cyanobacteria is amazingly diverse
and is a direct reflection of the unique biosynthetic capabilities of these organisms. L.
majuscula is clearly the most prominent producer of novel secondary metabolites
among these organisms. The hct gene cluster adds to the growing repertoire of
cyanobacterial hybrid PKS-NRPS pathways and contains several novel elements. An
interesting feature of this cluster is the juxtaposition of two multimodule ORF 's with
identical organization suggesting that it may have arisen through a gene duplication
event. Sequence alignment of HctE and HctF indicate >90% identity in certain
regions. Analysis of the putative hct gene cluster has revealed novel motifs such as
the presence of an embedded KR domain in an NRPS module, which is likely
involved in reducing an 0-IVA precursor. This is the first report of the presence of a
KR domain in an NRPS module from a cyanobacterial gene cluster, to the best of our
knowledge. Other notable features include an acyl-ACP synthetase, a putative
halogenase, two P450 monooxygenases and the presence of an IS element within the
cluster. Another striking feature of this gene cluster is the presence of homologs of
recognizable genetic elements such as JamA from the j amaicamide A gene cluster and
BarB 1 from the barbamide gene cluster that are found in new combinations (HctA and
HctB). The isolation of each new cluster has illustrated the remarkable versatility of
cyanobacterial biosynthetic machinery and provides the foundation for the harnessing
the activity of unique tailoring enzymes. The continued discovery of new molecules
125
from these metabolically talented organisms will provide a bountiful source of novel
bioactive natural products that may be developed as potential therapeutics.
Additionally, efforts focused at dissecting the biosynthesis of these metabolites at the
molecular genetic level along with whole-genome sequencing of several cyanobacteria
will enable us to fully exploit the biochemical diversity of these organisms.
126
EXPERIMENTAL
Bacterial strains and culture conditions
Escherichia coli strain DH1 OB was routinely used in this study as a host for
DNA cloning and sequencing while E. coli strain EPI 300 was used as a host for
construction of the fosmid library and amplification of fosmid clones. E. coli DH1OB
containing pGEM-T Easy, pBluescript or pGEM3ZF(+) was grown at 37°C overnight
in Luria-Bertani medium (LB) with a final concentration of ampicillin at 100 pg!ml.
E. coli EPI 300 harboring fosmid clones were grown at 37°C in LB medium with
chloramphenicol at a final concentration of 12 tg/ml. Lyngbya majuscula JHB was
collected from Hector Bay in 1996 and is maintained as a unigal culture in our
laboratory (Marquez et al., 2002).
DNA isolation and construction of fosmid library
DNA from Lyngbya majuscula JHB for PCR amplification was obtained using
DNeasy Plant Mini Kit (Qiagen, Valencia, CA). High molecular weight DNA for
construction of the library from L. majuscula JHB was extracted using phenol
followed by purification with Genomic Tip Kit (Qiagen, Valencia, CA) (Chapter.II).
The high molecular weight DNA was size selected (-' 40 kb), end repaired, and ligated
into the copy control fosmid vector pCC1FOS according to instructions provided with
the CopyControl Fosmid Library Production Kit (Epicentre, Madison, WI). Plasmid
DNA isolation was carried out using commercially available kits (Qiagen, Valencia,
CA). Other standard DNA manipulations such as restriction digests, ligations and
transformations were carried out using standard methods (Sambrook et al., 1989).
Library screening, labeling of probes, detection and Southern analysis were performed
according to the protocols provided with the DIG High Prime DNA Labeling and
Detection Starter Kit II (Roche Molecular Biochemicals, Manheim, Germany).
Prehybridization and hybridization of probes were carried out at 42°C and stringency
washes were carried out at 55°C.
127
PCR cloning of heterocyclization-adenylation domain probe
PCR amplification of probe fragments used to screen the genomic library in
this study was carried out using Taq DNA-polymerase (Invitrogen, Carlsbad, CA)
using manufacturer's recommendation regarding template and primer concentrations
in an EppendorfMastercycler. The following thermocycler conditions were used for
amplification: denaturation at 94°C for 30 s, annealing at 48°C for 30 s, extension at
72°C for 1 mm for 30 cycles. Degenerate primers were designed based on conserved
domains found in the heterocyclization-adenylation domain regions (HetForward-5'-
CCG GAT AAG CGA CAT GAA CCN TTY CCN YTN A-3' and AdenReverse-5'CAT CAG GGC GGG CAC NSW RTT CCA-3'). These primers were used to
amplify an approximately 1.7 kb fragment. The PCR product was purified using a
commercially available kit (Qiagen, Valencia, CA), cloned into pGEM-T Easy
(Promega, Madison, WI) and unique clones were sequenced. Amplification of an
approximately 700 bp region of the ketosynthase domain was carried out using
previously designed primers KS1Up 5'-MGI GAR GCI HWI SMI ATG GAY CCI
CAR CAl MG-3' and KSD1 5'- GGR TCI CCI ART SWI GTI CCR TG-3'(Beyer et
al., 1999). All primers were obtained from Tnvitrogen (Carlsbad, CA).
Sequence analysis of fosmids
Sequencing of fosmids pHctl and pHctII was carried out using shotgun
cloning. Fosmid DNA was subjected by partial digest using AluI or Sau3AI (0.100.25 units/.xg of fosmid DNA for 1 hour). The partially digested fosmid DNA was
then separated on a 1% agarose gel and fragments of size 1 kb-2. 5 kb were selected.
The AluI fragments were ligated into pGEM3Zf-(+) treated with SmaTJCIAP (calf
intestinal alkaline phosphatase) and Sau3AI fragments were cloned into pBluescript
that was digested with BamHT and ClAP-treated and random subclones were selected
for sequencing reactions. Sequencing was carried out using Big Dye ITT terminator
cycle sequencing (PE biosystems) at the Central Services Lab, Center for Gene
Research and Biotechnology, Oregon State University. Resulting gaps in the
sequences were filled using primer walking with sequence specific primers. The
128
sequences were edited and assembled using VectorNTl's ContigAssembly software
program (InforMax mc, Frederick, MD). Analyses of DNA and protein sequences
were carried out using the NCBI (National Center for Biotechnology Information)
BLAST server. All alignments were conducted using ClustalW (Thompson et aL,
1994).
Cloning, expression and purification of adenylation domains from HCtEO..1VA and
HctEc adenylation domains
The adenylation domains of HctE were amplified by PCR using Vent® DNA
polymerase (New England Biolabs) according to manufacturer's recommendations.
The following primers (5 'tail in italics; restriction digest sites in bold) HCtEAOIVA-
Forward3-5'GGA ATT CCA TAT GGA AAC TGA CAA CTT OAT CGA C 3' and
HctEAOWA-Reverse3-5' CCG CTC GAG GGA AGA CAG ATT AAA AAC AGG 3'
were used to amplify the adenylation domain from module 2 HctE (amino acids 787-
1391) and the primers HctEAc-Forward3-5'GGA ATTCCA TAT GTT CCA GAG
TTA CAC AGA TTA T 3' and HctEAc-Reverse3-5' CCG CTC GAG ATC AAA
TTC TGA CAG TGG TAA were used to amplify the adenylation domain of HctE3
(amino acid 2450-2956). The PCR products were cloned in-frame with a carboxyterminal 6xHis fusion at the NdeI and XhoI sites of the pET2Ob(+) expression vector
(Novagen). Both constructs were verified by sequencing. Overexpression of proteins
was carried out in E. coli BL21 (DE3) and both proteins were purified in an identical
manner. Overnight cultures expressing HctE2 A-domain or HctE3 A-domain were
diluted 1:100 in 1 liter of LB-broth containing 100 g/m1 ampicillin and grown at
18°C for about 12-18 hours without IPTG induction. Cells were harvested by
centrifugation, resuspended in lysis buffer (20 mM Tris-HC1 {pH8], 300 mM NaC1
and 20 mM imidazole) and lysed by sonication for six 10 s bursts at 75 mAmps using
a UPC 2000U sonicator (Ultrasonic Power Corporation, Freeport, IL). Recombinant
proteins were purified using nickel chelate chromatography (Qiagen, Valencia, CA).
Protein lysates were incubated with Ni-agarose beads for 2 hours at 4°C. The protein-
129
Ni-agarose slurry was then washed three times with wash buffer (20 mM Tris-HC1
[p11=8], 300 mM NaC1 and 40 mM imidazole) and proteins were eluted with elution
buffer (20 mM Tri-HC1 [pH=8], 300 mM NaC1 and 250 mM imidazole). Purified
proteins were dialyzed overnight at 4°C against storage buffer (50 mM Tris-HC1
[p11=8], 1 mlvi EDTA [p11=8], 100 mlvi NaC1, 10 mlvi MgC12, 1 mlvi DTT and 10%
glycerol) using Spectra/P or dialysis membrane with a molecular weight cutoff of
12,000-14,000 Da (Spectrum Labs mc, Rancho Dominguez, CA). The dialyzed
proteins were aliquoted, flash frozen in liquid nitrogen and stored at -80°C. Protein
concentration was determined by Bradford protein assay and protein purity was
assessed by analysis of protein lysates on a 12% SDS-PAGE followed by staining
with Coomassie blue staining.
Western blot analysis
Western blot analysis was carried out according to previously described
methods (Sambrook et al., 1989). Proteins were transferred onto nitrocellulose
membrane (Immobilon P, Millipore, Massachusetts) using Trans-Blot Electrophoretic
Transfer Cell (Biorad, California) according to manufacturer's instruction
accompanying the transfer unit. The membrane was first blocked in 5% nonfat dry
milk in TTBS (20 mM Tris, 137 mM NaC1, 0.1% Tween, pH 7.6) for one hour. The
membrane was the incubated with a-His antibody, the primary antibody (1:1000) in
1% non-fat dry milk /TTBS for 1 hour. After three washes in TTBS (20 minutes
each), the membrane was incubated with horseradish peroxidase (HRP) conjugate
secondary antibody (1:7500) for one hour. The membrane was washed four times in
TTBS (20 minutes each) and chemiluminiscent detection using standard ECL kit was
used to visualize protein bands.
130
Substrate-dependent ATP-pyrophosphate [32PPiI exchange assay
All amino acid substrates used in this assay were obtained from Sigma (St
Louis, MO) or Lancaster (Windham, NH). 2-oxo-3-hydroxy isovaleric acid and DHiv
were synthesized using previously described methods (Hanson et al., 1990; Luesch et
al., 2000b). The substrate-dependent ATP-PPi exchange assay was performed by
incubating 2 M of HCtEOWA or HctEc in reaction buffer (50 mM Tris-HC1 [pH=8],
100 mM NaC1, 10 mM MgC12, 1 mM DTT) containing 0.5 tCi tetrasodium [32P]
pyrophosphate (NEN-Perkin Elmer, Boston, MA) and 2 mM of the respective amino
acid or oxo-derivative for 1 hour at 28°C. The reaction was quenched by the addition
of stop-mix (500 il of 1.2% (w/v) activated charcoal, 0.1 M tetrasodium
pyrophosphate and 0.35 M perchioric acid). The charcoal pellet was pelleted and
washed three times with wash buffer (0.1 M tetrasodium pyrophosphate and 0.35 M
perchioric acid) and resuspended in 500 d of water. The bound radioactivity was
determined using a Packard TriCarb 2900 liquid scintillation counter.
Cloning, expression and purification of HctA
The adenylation domains of HctE were amplified by PCR using Vent® DNA
polymerase (New England Biolabs) according to manufacturer's recommendations.
The following primers (5 'tail in italics; restriction digest sites in bold) were used for
the amplification of HctA. HctAForward: 5' GGA ATT CCA TAT GAA TAC TAA
GAA AAA ATT TAC CTC T 3' and HctAReverse: 5' CCG CTC GAG CCC CAG
ATG CGC CCC ATT TTT 3'. The PCR products were cloned in-frame with a
carboxy-terminal 6xHis fusion at the NdeI and XhoI sites of the pET2Ob(+) expression
vector (Novagen) and verified by sequencing. Overexpression of proteins was carried
out in E. coli BL21 (DE3) and overnight cultures expressing HctA were diluted 1:100
in 1 liter of LB-broth containing 100 p.g/ml ampicillin and grown at 25°C until
0D600
= 0.4 before induction with 0.4 mM IPTG. Protein purification was carried out as
described above.
131
Accession number
The nucleotide sequence of the hectochiorin biosynthetic gene described in this
work has been submitted to GenBank under the accession number AY974560.
132
REFERENCES
Andersen, J. F. and Hutchinson, C. R. (1992). Characterization of Saccharopolyspora
erythraea cytochrome P-450 genes and enzymes, including 6-deoxyerythronolide B
hydroxylase. Journal of Bacteriology 174, 725-735.
Bancroft, I. and Wolk, C. P. (1989). Characterization of an insertion sequence (1S891)
of novel structure from the cyanobacterium Anabaena sp. strain M-1 31. Journal of
Bacteriology 171, 5949-5954.
Becker, J. E., Moore, R. E., and Moore, B. S. (2004). Cloning,sequencing,and
biochemical characterization of the nostocyclopeptide biosynthetic gene
cluster:molecular basis for imine macrocyclization. Gene 325, 35-42.
Beyer, S., Kunze, B., Silakowski, B., and Muller, R. (1999). Metabolic diversity in
myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene
cluster of Stigmatella aurantiaca Sg a15 and a combined polyketide-Qoly)peptide
gene cluster from the epothilone producing strain Sorangium cellulosum So ce9O.
Biochimica Et Biophysica Acta 1445, 185-195.
Burja, A. M., Banaigs, B., Abou-Mansour, E., Burgess, J. G., and Wright, P. C.
(2001). Marine cyanobacteria: A prolific source of natural products. Tetrahedron 57,
9347-9377.
Censini, S., Lange, C., Xiang, Z., Crabtree, J. E., Ghiara, P., Borodovsky, M.,
Rappuoli, R., and Covacci, A. (1996). cag, a pathogenicity island of Helicobacter
pylon, encodes type I-specific and disease-associated virulence factors. Proceedings of
the National Academy of Sciences of the United States of America 93, 14648-14653.
Cetusic, J. R. P., Green, F. R., Graupner, P. R., and Oliver, M. P. (2002). Total
Synthesis of Hectochiorin. Organic Letters 4, 1307-13 10.
Challis, G. L., Ravel, J., and Townsend, C. A. (2000). Predictive, structure-based
model of amino acid recognition by nonribosomal peptide synthetase adenylation
domains. Chemistry & Biology 7,211-224.
Chang, Z., Flatt, P., Gerwick, W. H., Nguyen, V. A., Willis, C. L., and Sherman, D. H.
(2002). The barbamide biosynthetic gene cluster: a novel marine cyanobacterial
system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS)
origin involving an unusual trichioroleucyl starter unit. Gene 296, 235-247.
Chang, Z., Sitachitta, N., Rossi, J. V., Roberts, M. A., Flatt, P. M., Jia, J., Sherman, D.
H., and Gerwick, W. H. (2004). Biosynthetic pathway and gene cluster analysis of
curacin A, an antitubulin natural product from the tropical marine cyanobacterium
Lyngbya majuscula. Journal of Natural Products 67, 1356-1367.
133
Chen, H., Hubbard, B. K., O'Connor, S. E., and Walsh, C. T. (2002). Formation of
beta-hydroxy histidine in the biosynthesis of nikkomycin antibiotics. Chemistry &
Biology 9, 103-112.
Chen, H., Thomas, M. G., O'Connor, S. E., Hubbard, B. K., Burkart, M. D., and
Walsh, C. T. (2001). Aminoacyl-S-enzyme intermediates in beta-hydroxylations and
alpha,beta- desaturations of amino acids in peptide antibiotics. Biochemistry 40,
1165 1-9.
Chen, H. and Walsh, C. T. (2001). Coumarin formation in novobiocin biosynthesis:
beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450
NovI. Chemistry & Biology 8, 301-3 12.
Christiansen, G., Fastner, J., Erhard, M., Borner, T., and Dittmann, E. (2003).
Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation. J
Bacteriol 185, 564-72.
Donadio, S. and Stayer, M. J. (1993). 1S1136, an Insertion Element in the
Erythromycin Gene Cluster of Saccharopolyspora erythraea. Gene 126, 147-15 1.
Du, L., Chen, M., Sanchez, C., and Shen, B. (2000a). An oxidation domain in the
Bimill non-ribosomal peptide synthetase probably catalyzing thiazole formation in the
biosynthesis of the anti- tumor drug bleomycin in Streptomyces verticillus
ATCC15003. FEMS Microbiology Letters 189, 171-175.
Du, L., Sanchez, C., Chen, M., Edwards, D. J., and Shen, B. (2000b). The biosynthetic
gene cluster for the antitumor drug bleomycin from Streptomyces verticillus
ATCC 15003 supporting functional interactions between nonribosomal peptide
synthetases and a polyketide synthase. Chemistry & Biology 7, 623-642.
Edwards, D. J. and Gerwick, W. H. (2004). Lyngbyatoxin biosynthesis: sequence of
biosynthetic gene cluster and identification of a novel aromatic prenyltransferase.
Journal of American Chemical Society 126, 11432-11433.
Edwards, D. J., Marquez, B. L., Nogle, L. M., McPhail, K., Goeger, D. E., Roberts, M.
A., and Gerwick, W. H. (2004). Structure and biosynthesis of the jamaicamides, new
mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya
majuscula. Chemistry & Biology 11, 8 17-833.
Ehling-Schulz, M., Vukov, N., Schulz, A., Shaheen, R., Andersson, M., Martlbauer,
E., and Scherer, S. (2005). Identification and partial characterization of the
nom-ibosomal peptide synthetase gene responsible for cereulide production in emetic
Bacillus cereus. Applied and Environmental Microbiology 71, 105-113.
Finking, R. and Marahiel, M. (2004). Biosynthesis of nonribosomal peptides. Annual
Review of Microbiology 58, 453-488.
134
Gehring, A. M., Mori, I., Perry, R. D., and Walsh, C. T. (1998). The nonribosomal
peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of
yersiniabactin, an iron-chelating virulence factor of Yersinia pestis. Biochemistry 37,
11637-11650.
Gerwick,W.H., Tan,L., and Sitachitta,N. (2001). Nitrogen-containing metabolites from
marine cyanobacteria. In The Alkaloids, G.A.Cordell, ed. (San Diego: Academic
Press), pp. 75-184.
Guenzi, B., Galli, G., Grgurina, I., Gross, D. C., and Grandi, G. (1998).
Characterization of the syringomycin synthetase gene cluster - A link between
prokaryotic and eukaryotic peptide synthetases. Journal of Biological Chemistry 273,
32857-32863.
Hanson, R. L., Singh, J., Kissick, T. P., Patel, R. N., Szarka, L. J., and Mueller, R. H.
(1990). Synthesis of L-Hydroxyvaline from aKeto-[3hydroxy isovalerate using
leucine dehydrogenase from Bacillus sp. Bioorganic Chemistry 18, 116-130.
Haydock, S. F., Aparicio, J. F., Molnar, I., Schwecke, T., Khaw, L. E., Konig, A.,
Marsden, A. F., Galloway, I. S., Staunton, J., and Leadlay, P. F. (1995). Divergent
sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl
carrier protein transacylase domains in modular polyketide synthases. Febs Letters
374, 246-248.
Kershaw, N. J., Mukherji, M., MacKinnon, C. H., Claridge, T. D., Odell, B.,
Wierzbicki, A. S., Lloyd, M. D., and Schofield, C. J. (2001). Studies on phytanoylCoA 2-hydroxylase and synthesis of phytanoyl-coenzyme A. Bioorganic & Medicinal
Chemistry 11, 2545-2548.
Kuse, M., Franz, T., Koga, K., Suwan, S., Isobe, M., Agata, N., and Ohta, M. (2000).
High incorporation of L-amino acids to cereulide, an emetic toxin from Bacillus
cereus. Bioorganic & Medicinal Chemistry Letters 10, 735-739.
Luesch, H., Yoshida, W. Y., Moore, R. E., and Paul, V. J. (2000a). Isolation and
structure of the cytotoxin lyngbyabellin B and absolute configuration of lyngbyapeptin
A from the marine cyanobacterium Lyngbya majuscula. Journal of Natural Products
63, 1437-1439.
Luesch, H., Yoshida, W. Y., Moore, R. B., Paul, V. J., and Mooberry, S. L. (2000b).
Isolation, structure determination, and biological activity of Lyngbyabellin A from the
marine cyanobacterium Lyngbya majuscula. Journal of Natural Products 63, 611-615.
Mahillon, J. and Chandler, M. (1998). Insertion sequences. Microbiology and
Molecular Biology Reviews 62, 725-774.
135
Mahillon, J., Leonard, C., and Chandler, M. (1999). IS elements as constituents of
bacterial genomes. Research in Microbiology 150, 675-687.
Marquez, B. L., Watts, K. S., Yokochi, A., Roberts, M. A., Verdier-Pinard, P.,
Jimenez, J. I., Hamel, E., Scheuer, P. J., and Gerwick, W. H. (2002). Structure and
absolute stereochemistry of hectochlorin, a potent stimulator of actin assembly.
Journal of Natural Products 65, 866-871.
Milligan, K. E., Marquez, B. L., Williamson, R. T., and Gerwick, W. H. (2000).
Lyngbyabellin B, a toxic and antifungal secondary metabolite from the marine
cyanobacterium Lyngbya majuscula. Journal of Natural Products 63, 1440-3.
Moffitt, M. C. and Neilan, B. A. (2004). Characterization of the nodularin synthetase
gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins.
Applied and Environmental Microbiology 70, 6353-6362.
Molnar, I., Schupp, T., Ono, M., Zirkie, R., Milnamow, M., Nowak-Thompson, B.,
Engel, N., Toupet, C., Stratmann, A., Cyr, D. D., Gorlach, J., Mayo, J. M., Hu, A.,
Goff, S., Schmid, J., and Ligon, J. M. (2000). The biosynthetic gene cluster for the
microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So
ce9O. Chemistry & Biology 7, 97-109.
Mukherji, M., Chien, W., Kershaw, N. J., Clifton, I. J., Schofield, C. J., Wierzbicki, A.
S., and Lloyd, M. D. (2001). Structure-function analysis of phytanoyl-CoA 2hydroxylase mutations causing Refsum's disease. Human Molecular Genetics 10,
1971-1982.
Murai, N., Kamata, H., Nagashima, Y., Yagisawa, H., and Hirata, H. (1995). A novel
insertion sequence (IS)-like element of the thermophilic bacterium PS3 promotes
expression of the alanine carrier protein-encoding gene. Gene 163, 103-107.
Neilan, B. A., Dittmann, E., Rouhiainen, L., Bass, R. A., Schaub, V., Sivonen, K., and
Borner, T. (1999). Nonribosomal peptide synthesis and toxigenicity of cyanobacteria.
Journal of Bacteriology 181, 4089-4097.
Newman, D. J., Cragg, G. M., and Snader, K. M. (2003). Natural products as sources
of new drugs over the period 1981-2002. Journal of Natural Products 66, 1022-1037.
Paitan, Y., On, E., Ron, E. Z., and Rosenberg, E. (1999a). Cloning and
characterization of a Myxococcus xanthus cytochrome P-450 hydroxylase required for
biosynthesis of the polyketide antibiotic TA. Gene 228, 147-53.
Paitan, Y., On, E., Ron, E. Z., and Rosenberg, E. (1999b). Genetic and functional
analysis of genes required for the post- modification of the polyketide antibiotic TA of
Myxococcus xanthus. Microbiology 145, 3059-3067.
136
Quadri, L. B. N., Keating, T. A., Pate!, H. M., and Walsh, C. 1. (1999). Assembly of
the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin: In vitro
reconstitution of aryl-4,2-bisthiazoline synthetase activity from PchD, PPchE, and
PchF. Biochemistry 38, 14941-14954.
Reid, R., Piagentini, M., Rodriguez, E., Ashley, G., Viswanathan, N., Carney, J.,
Santi, D. V., Hutchinson, C. R., and McDaniel, R. (2003). A mode! of structure and
catalysis for ketoreductase domains in modular polyketide synthases. Biochemistry 42,
72-79.
Rose, T. M., Schultz, E. R., Henikoff, J. G., Pietrokovski, S., McCallum, C. M., and
Henikoff, S. (1998). Consensus-degenerate hybrid oligonucleotide primers for
amplification of distantly related sequences. Nucleic Acids Research 26, 1628-35.
Sambrook,J., Fritsch,E.F., and Maniatis,T. (1989). Molecular Cloning: A Laboratory
Manual. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).
Schneider, T. L., Shen, B., and Walsh, C. T. (2003). Oxidase domains in epothiolone
and bleomycin biosynthesis: thiazoline to thiazole oxidation during chain elongation.
Biochemistry 42, 9722-9730.
Schwarzer, D., Finking, R., and Marahiel, M. A. (2003). Nonribosomal peptides: from
genes to products. Natural Product Reports 20, 275-287.
Silakowski, B., Schairer, H. U., Ehret, H., Kunze, B., Weinig, S., Nordsiek, G.,
Brandt, P., Blocker, H., Hofle, G., Beyer, S., and Muller, R. (1999). New lessons for
combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene
cluster of Stigmatella aurantiaca DW413-1. Journal of Biological Chemistry 274,
37391-37399.
Sitachitta, N., Rossi, J., Roberts, M. A., Gerwick, W. H., Fletcher, M. D., and Willis,
C. L. (1998). Biosynthesis of the marine cyanobacterial metabolite barbamide. 1.
Origin of the trichioromethyl group. Journal of American Chemical Society 120,
7131-7132.
Sone, H., Kondo, T., Kiruyu, M., Ishiwata, H., Ojika, M., and Yamada, K. (1995).
Dolabellin, a Cytotoxic Bisthiazole Metabolite from the Sea Hare Dolabella
auricularia: Structural Determination and Synthesis. Journal of Organic Chemistry 60,
4774-4781.
Stacheihaus, T. and Marahiel, M. A. (1995). Modular structure of peptide synthetases
revealed by dissection of the multifunctional enzyme GrsA. Journal of Biological
Chemistry 270, 6163-6169.
137
Stachelhaus, T., Mootz, H. D., and Marahiel, M. A. (1999). The specificity-conferring
code of adenylation domains in nonribosomal peptide synthetases. Chemistry &
Biology 6, 493-505.
Steffensky, M., Muhlenweg, A., Wang, Z. X., Li, S. M., and Heide, L. (2000).
Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides
NCIB 11891. Antimicrobial Agents and Chemotherapy 44, 1214-1222.
Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids
Research 22, 4673-4680.
Tillett, D., Dittmann, E., Erhard, M., von Dohren, H., Bonier, T., and Neilan, B. A.
(2000). Structural organization of microcystin biosynthesis in Microcystis aeruginosa
PCC7806: an integrated peptide-polyketide synthetase system. Chemistry & Biology
7, 753-764.
Ullrich, M. and Bender, C. L. (1994). The biosynthetic gene cluster for coronamic
acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acidactivating enzymes and thioesterases. Journal of Bacteriology 176, 7574-7586.
Varon, M., Fuchs, N., Monosov, M., Tolchinsky, S., and Rosenberg, E. (1992).
Mutation and mapping of genes involved in production of the antibiotic TA in
Myxococcus xanthus. Antimicrobial Agents and Chemotherapy 36, 23 16-2321.
Wang, Z. X., Li, S. M., and Heide, L. (2000). Identification of the coumermycin A(1)
biosynthetic gene cluster of Streptomyces rishiriensis DSM 40489. Antimicrobial
Agents and Chemotherapy 44, 3040-3048.
Weissman, K. J. (2004). Polyketide biosynthesis: understanding and exploiting
modularity. Philosophical Transactions of the Royal Society of London Series AMath, Physics and Engineering Sciences 362, 267 1-2690.
Zhang, J. H., Quigley, N. B., and Gross, D. C. (1995). Analysis of the syrB and syrC
genes of Pseudomonas syringae pv. syringae indicates that syringomycin is
synthesized by a thiotemplate mechanism. Journal of Bacteriology 177, 4009-4020.
138
CHAPTER IV
EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF
ADENYLATION DOMAINS FROM THE CARMABIN BIOSYNTHETIC
GENE CLUSTER
INTRODUCTION
A Caribbean collection of the cyanobacterium, Lyngbya majuscula (designated
as strain 1 9L) was an extraordinarily rich source of bioactive metabolites and yielded
several novel metabolites including barbamide (53) (Orjala and Gerwick, 1996), the
curacins (curacin A 14) (Gerwick et al., 1994) and antillatoxin (25) (Orj ala et al.,
1995). The more polar lipid extract yielded minute quantities (' 1 mg) of a
lipopeptide, carmabin A (55) (Hooper et al., 1998). The cyanobacterium was
subsequently recollected and carmabin A was re-isolated along with the closely related
carmabin B (56) (Hooper et al., 1998). Carmabin A was the first L. majuscula-derived
metabolite to possess a terminal acetylene funcationality (Gerwick et al., 2001). The
producing organism has been maintained in stable culture in our laboratory for 12
years. Although carmabin A was initially isolated as an antiproliferative agent (IC50
6.58 tg/mL against MRC-5 embryonic lung cell), the re-isolated pure compound
proved to be inactive in the NC1-60 cell line assay (Hooper et al., 1998). A related
metabolite, dragonamide (57) has also been isolated from a Panamanian strain of L.
majuscula (Jiménez and Scheuer, 2001).
Interesting structural features of carmabin A, such as its terminal
functionalization, stimulated efforts to clone and identify the gene cluster involved in
its biosynthesis. A genomic DNA library of Lyngbya majuscula 19L was screened
with KS-domain probes amplified using previously described primers (Beyer et al.,
1999). This led to the identification of two overlapping cosmids, pLM32 and pLM23
as putative candidates for the carmabin biosynthetic gene cluster (car) (Z. Chang and
D.H. Sherman, unpublished). Sequence analyses of these two cosmids led to the
139
mapping of an approximately 42 kb region consisting of ten ORFs that may be
involved in the biosynthesis of carmabin A (Figure IV.1) (Z. Chang, J. Junyong and
D.H. Sherman, unpublished).
H
CH3
OCH3 ccI
H
OCH3
NS
H
Curacin A (14)
Barbamide (53)
Ant illatoxin (25)
0
0
0
Carmabin A (55)
-0CH3
0
Carmabin B (56)
OCH3
0
I
Dragonamide (57)
NCH3
'H
140
42
ORF
PKS
car AB
PKS
D
E
KS-AT-DH-MT-ER-KR-ACP
NRPS
G
NRPS
F
-
KS-AT-DH-MT-ER-KR-ACP
.
C-AN-PCP
C-AphNMt.PCP
I
NRPS
NRPS
H
I
C-ATY,-N-Mt-O.Mt-PCP-AmT Hydrolase!TE
CAN4.lt-PCP
2.8 kb
pLM32
pLM23
Figure IV. 1: Genetic architecture of the carmabin A biosynthetic gene cluster
(Z. Chang, J. Junyong, D. H. Sherman and W.H. Gerwick, unpublished).
Table IV.!: Deduced functions of open reading frames in the car gene
cluster
Protein
Size
Proposed function
(amino
acids)
CarA
--402
Acyl-ACP synthetase
CarB
321
Desaturase
CarC
100
ACP
CarD
2549
PKS
CarE
2575
PKS
CarF
1545
NRPS
CarG
1135
NRPS
CarH
1546
NRPS
Carl
2303
NIRPS
CarJ
293
Predicted
hydrolase/thioesterase
Sequence
similarity
(protein,
organism)
JamA,
L. ma] uscula
JamB,
L.majuscula
JamC,
L. majuscula
JamJ,
L.majuscula
JamJ,
L. majuscula
BarG,
L. ma] uscula
JamO,
L. majuscula
BarG,
L. majuscula
NcpB,
Nostoc ATCC
53789
Nostoc
punctforme
PCC 73102
%
Reference
Identity,
Similarity
96%,98%
95%,98%
95%, 98%
68%,80%
69%,81%
67%,81%
49%,68%
52%,67%
55%,71%
(Edwards et
al., 2004)
(Edwards et
al., 2004)
(Edwards et
al., 2004)
(Edwards et
al., 2004)
(Edwards et
al., 2004)
(Chang et al.,
2002)
(Edwards et
al., 2004)
(Chang et al.,
2002)
(Beckeretal.,
2004)
67%,78%
141
Complete sequence information for CarA is currently lacking and sequencing
of cosmid pLM32 is in progress to obtain this information. Additionally, sequencing
will also provide insights into the region directly upstream of CarA and thus enable
proper delineation of the car cluster.
Overall,
car
appears to be generally co-linear and fully consistent with the
structure of carmabin A (55). Based on their similarity to JamA-C proteins (Table
IV. 1), it is proposed that the CarA-C proteins comprise a system for the generation of
a hexynoic acid starter unit (Edwards et al., 2004). The starter unit is then expected to
undergo two rounds of ketide extension catalyzed by CarD and CarE, both of which
possess KS-AT-DH-MT-ER-KR-ACP domain organization. Next, the incorporation
of the four amino acids (N-methyl phenylalanine, alanine, N-methyl alanine and N-
methyl 0-methyl tyrosine) is most likely catalyzed by the proteins CarF-I (Table
IV.2). CarF and CarH contain embedded N-methyl transferase domains, while Carl
contains an N-methyl transferase domain along with an 0-methyl transferase domain
(Schwarzer et al., 2003). Finally, release of carmabin A (55) is likely catalyzed by the
TE encoded by CarJ.
142
Table IV.2: Substrate specificity of A-domains in car. Amino acid residues
involved in substrate recognition from adenylation domains of CarF, CarG,
CarH and Carl were analyzed using the method of Challis et al. and
Stacheihaus et al. The predicted amino acid specificity is completely
consistent with the observed amino acids of carmabin A (55).
A-domain
Binding pocket amino acids
Predicted
substrate
235
236
239
278
299
301
322
330
CarF
D
A
W
T
V
A
A
V
phenylalanine
CarG
D
L
F
N
N
A
L
T
alanme
CarH
D
L
F
N
N
A
L
T
alanine
Carl
D
A
S
T
V
A
A
V
tyrosine
In order to confirm the activities of the NRPS adenylation domains and to
provide further biochemical evidence for the involvement of this gene cluster in the
biosynthesis of carmabin A (55), we sought to overexpress the adenylation domains
and analyze them using the ATP-PPi exchange assay (Stachelhaus and Marahiel,
1995). Herein, the cloning, overexpression and biochemical characterization of two
adenylation domains from the carmabin gene cluster is reported.
143
RESULTS AND DISCUSSION
Cloning and overexpression of adenylation domains
Since methods for targeted gene disruption are not yet available in
L.
majuscula, overexpression and biochemical characterization of the adenylation
domains is important for establishing the role of car in the biosynthesis of carmabin A
(4). A similar method has been used for other L. majuscula derived biosynthetic gene
clusters (Chang et al., 2002; Edwards et al., 2004), and other cyanobacterial gene
clusters (Becker et al., 2004; Hoffmann et al., 2003).
Primers for the amplification of the A-domains from the car cluster were based
on the start site of the construct designed for the expression ofj amaicamide JamO
which has been successfully expressed (Edwards et al., 2004). The stop site of CarG
A domain was based on that of JamO construct, however the CarF, CarH and Carl Adomains had to be truncated because of embedded tailoring domains and
consequently, stop sites were designed downstream of the QVKRIG motif (A8 motif).
The adenylation domains were amplified, cloned into the pET2Ob(+) expression
vector with a C-terminal 6x His-tag and transformed into E. coli BL2 1 (DE3) cells for
expression. Optimal protein expression was obtained for CarG and Carl A-domains at
25°C and induction with 0.4 mM IPTG. Western blot analysis using a-His antibodies
was performed to confirm heterologous protein expression. However, protein
expression was not observed for the CarF and CarH proteins although expression was
examined under several temperature conditions (18°C, 22°C, 25°C and 30°C) and with
different IPTG concentrations (0.2 mM, 0.4mM) to optimize expression and yield.
Our initial goal was to characterize all four A-domains of the car cluster, but
we succeeded in overexpressing and biochemically characterizing only two, CarG and
Carl A-domains. In general, functional expression of adenylation domains has proven
to be challenging. Complete absence of protein expression, very low expression levels
or inactivity of expressed proteins is frequently encountered. In the case of
chloroeremomycin biosynthetic gene cluster, although low levels of protein expression
for CepA adenylation domain was observed, the protein was inactive in subsequent
144
ATP-PPi exchange assays and lack of activity was attributed to misfolding or partial
unfolding during purification (Trauger and Walsh, 2000). The expression of NRPS
domains with chaperone proteins such as GroES/EL has been shown to facilitate
functional expression of these domains (Symmank et al., 1999). However, expression
in the presence of such chaperones does not always result in the production of active
protein and this has been observed for chioroeremomycin A-domains (Trauger and
Walsh, 2000) and curacin CurF A domain (P. Flatt, personal communication).
Another important factor impacting the functional expression of these proteins
is the host organism. For instance, expression of adenylation domains from the
baihimycin biosynthetic gene cluster could not be detected in E. coli whereas
expression of these proteins was readily achieved in a Streptomyces host (Recktenwald
et al., 2002). Similarly, several other proteins have been successfully expressed in a
functional form in a Streptomyces sp. heterologous host (Malpartida and Hopwood,
1984; Tang et al., 2000). However, preliminary investigations conducted in our
laboratory by P. Flatt and G. Zeller revealed that cyanobacterial genes demonstrated a
high level of instability in Streptomyces host systems and often undergo
rearrangements. Thus, this organism was not feasible for the expression of
cyanobacterial genes. Studies have also shown that protein expression is more
efficient in heterologous hosts that are more closely related to the parental strain. For
example, in the case of epothilone biosynthesis, heterologous expression in
Myxococcus xanthus resulted in higher yields of epothilone when compared to a
Streptomyces host (Julien and Shah, 2002; Tang et al., 2000). At present, Nostoc
punctiforme ATCC 29133 is being considered an as alternate host for heterologous
expression. The genome of this strain has been sequenced and several molecular tools
have been developed that allow for the design and development of genetic
manipulation experiments (Meeks et al., 2001; Wong and Meeks, 2001)
The heterologous expression of functional cyanobacterial proteins poses a
major challenge and the development of new methods and strategies is an important
goal of current research. Additionally, dissection of these modular systems at the
145
biochemical and genetic levels will enable us to accomplish the heterologous
expression of functional proteins.
Biochemical characterization of the adenylation domains
CarG and Carl A-domains were purified by using Ni2taffinity
chromatography (Figure IV.2A-B). Substrate specificity of the two adenylation
domains was demonstrated through the ATP-PPi exchange assay (Stacheihaus and
Marahiel, 1995).
A
60
Marker
(kDa)
Marker
(kDa)
B
CarG-AIa
60 kDa
Carl-Tyr
- kDa
55
50
20
20
Figure IV.2: Overexpression of CarG (A) (60 kDa) and Carl (B) (49
kDa) adenylation domains.
146
The relative activities of the various amino acid substrates in the ATP-PPi
exchange assay are displayed in Figures IV.3A and IV.3B. CarG A-domain activated
L-alanine to the highest level (=100%) and L-valine was activated to about 60%.
Other amino acids tested were activated to a much lower level ( 5%) (Figure IV.3A).
Analysis of the binding pocket amino acids in CarG A-domain (Table IV.2) revealed
that the residues were identical to those present in JamO A-domain, which has also
been shown to activate L-alanine to the highest level (Edwards et al., 2004). Other Lalanine activating A-domains with identical binding pocket amino acids include
bleomycin Bim VII (Du et al., 2000), microcystin Mcy A-2 (Tillett et al., 2000) and
myxalamid Mxa A (Silakowski et al., 2001).
In the case of the Carl A-domain, L-tyro sine was activated to the highest level
(= 100%) and L-phenyalanine was activated up to 30%, while other amino acids tested,
were activated to a much lesser extent (Figure IV.3B). The binding pocket residues in
Carl (Table IV.2) are identical to those present in nostocyclopeptide NcpAl tyrosine
activating domain (Becker et al., 2004).
Biochemical characterization of these two adenylation domains provides
further evidence to support the involvement of car in the biosynthesis of carmabin A.
147
A
120
100
80
C)
w
>
60
40
20
0
L-AIa
L-VaI
L-His
L-Iso
L-Trp
L.Ser
L-GIn
L.Tyr
L-GIy
L-Phe
L-Iso
L-GIn
L-Ser
L-His
B
120
100
80
>
C)
>
60
40
20
0
Figure IV.3: Results for the ATP-PPi exhange assay for CarG A-domain (A)
and Carl A-domain (B).
Abbreviations: L-Ala, alanine; L-Val, valine; L-His, hisitidine, L-Ile,
isoleucine; L-Trp, tryptophan; L-Ser, serine, L-Gln, glutamine; L-Tyr,
tyrosine, L-Phe, phenylalanine.
EXPERIMENTAL
Cloning, expression and purification of adenylation domains from car
The adenylation domains of car were amplified by PCR using Vent® DNA
polymerase (New England Biolabs) according to manufacturer's recommendations.
The following primers (5 'tail in italics; restriction digest sites in bold) were used for
PCR amplification of individual A domains.
CarF-Forward 5' GGA ATTCCA TAT GGT AAC AGC ACT ACC ATT AAT G 3';
CarF-Reverse 5' CCG CTC GAG TGG TAC TAT ATA TGC TAC TAG ACG 3';
CarG- Forward-5'GGA ATTCCA TAT GTT GCA GTT GCC ACT AAT GAA A 3';
CarG-Reverse 5' CCG CTC GAG TTG TGT CTC CGT CTC AGG 3';
CarH-Forward 5' GGA ATTCCA TAT GAG CCA GTT GCC ATT AAT GAC 3';
CarH-Reverse 5' CCG CTC GAG TCC AGG CTT ATC CTC TCT G 3';
Carl-Forward 5' GGA ATTCCA TAT GCC GAT TAG CCA GTT GCC A 3';
Carl-Reverse 5' CCG CTC GAG CCC TGG AGT ATC TTC CTT GT 3'.
The PCR products were cloned in-frame with a carboxy-terminal 6xHis fusion
at the NdeI and XhoI sites of the pET2Ob(+) expression vector (Novagen). Intially, 50
mL of E coli BL2 1 (DE3) containing each construct were tested for protein expression
under various temperature conditions (18°C, 22°C, 25°C and 3 7°C) and
concentrations of IPTG (0.2mM and 0.4mM). Protein expression was optimized only
for CarG and Carl A-domains. Both CarG and Carl A-domain constructs were
verified by sequencing. Overexpression of proteins was carried out in E.
coli
BL21
(DE3) and both proteins were purified in an identical manner. Overnight cultures
expressing CarG A-domain or Carl A-domain were diluted 1:100 in 1 liter of LBbroth containing 100 jig/ml ampicillin and grown at 25°C till
0D600
= 0.4. Cultures
were then induced with 0.4 mM IPTG and allowed to grow for an additional eight
hours. Cells were harvested by centrifugation, resuspended in lysis buffer (20 mM
Tris-HC1 {pH=8], 300 mM NaC1 and 20 mM imidazole) and lysed by sonication for
six 10 s bursts at 75 mAmps using a UPC 2000U sonicator (Ultrasonic Power
Corporation, Freeport, IL). Recombinant proteins were purified using nickel chelate
chromatography (Qiagen, Valencia, CA). Protein lysates were incubated with Niagarose beads for 2 hours at 4°C. The protein-Ni-agarose slurry was then washed
three times with wash buffer (20 mM Tris-HC1 [pH=8], 300 mM NaC1 and 40 mM
imidazole) and proteins were eluted with elution buffer (20 mM Tri-HC1 [pH=8], 300
mM NaC1 and 250 mM imidazole). Purified proteins were dialyzed overnight at 4°C
against storage buffer (50 mM Tris-HC1 [pH=8], 1 mM EDTA [pH8], 100 mM NaC1,
10 mM MgC12, 1 mM DTT and 10% glycerol) using Spectra/Por dialysis membrane
with a molecular weight cutoff of 12,000-14,000 Da (Spectrum Labs mc, Rancho
Dominguez, CA). The dialyzed proteins were aliquoted, flash frozen in liquid
nitrogen and stored at -80°C. Protein concentration was determined by Bradford
protein assay and protein purity was assessed by analysis of protein lysates on a 12%
SDS-PAGE followed by staining with Coomassie blue staining.
Substrate-dependent ATP-pyrophosphate [32PPiI exchange assay
All amino acid substrates used in this assay were obtained from Sigma (St
Louis, MO) or Lancaster (Windham, NH). The substrate-dependent ATP-PPi
exchange assay was performed by incubating 2 mM of CarG or Carl A domains in
reaction buffer (50 mM Tris-HC1 [pH=8], 100 mM NaC1, 10 mM MgC12, 1 mM DTT)
containing 0.5 tCi tetrasodium
[32
P] pyrophosphate (NEN-Perkin Elmer, Boston,
MA) and 2 mM of each amino acid for 1 hour at 28°C. The reaction was quenched by
the addition of stop-mix (500 tl of 1.2% (w/v) activated charcoal, 0.1 M tetrasodium
pyrophosphate and 0.35 M perchloric acid). The charcoal pellet was pelleted and
washed twice with wash buffer (0.1 M tetrasodium pyrophosphate and 0.35 M
perchioric acid) and resuspended in 500 tl of water. The bound radioactivity was
determined using a Packard TriCarb 2900 liquid scintillation counter.
150
REFERENCES
Becker, J. E., Moore, R. E., and Moore, B. S. (2004). Cloning,sequencing,and
biochemical characterization of the nostocyclopeptide biosynthetic gene
cluster:molecular basis for imine macrocyclization. Gene 325, 35-42.
Beyer, S., Kunze, B., Silakowski, B., and Muller, R. (1999). Metabolic diversity in
myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene
cluster of Stigmatella aurantiaca Sg al 5 and a combined polyketide-(poly)peptide
gene cluster from the epothilone producing strain Sorangium cellulosum So ce9O.
Biochimica Et Biophysica Acta 1445, 185-195.
Chang, Z., Flatt, P., Gerwick, W. H., Nguyen, V. A., Willis, C. L., and Shemian, D. H.
(2002). The barbamide biosynthetic gene cluster: a novel marine cyanobacterial
system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS)
origin involving an unusual trichioroleucyl starter unit. Gene 296, 23 5-247.
Du, L., Sanchez, C., Chen, M., Edwards, D. J., and Shen, B. (2000). The biosynthetic
gene cluster for the antitumor drug bleomycin from Streptomyces verticillus
ATCC 15003 supporting functional interactions between nonribosomal peptide
synthetases and a polyketide synthase. Chemistry & Biology 7, 623-642.
Edwards, D. J., Marquez, B. L., Nogle, L. M., McPhail, K., Goeger, D. B., Roberts, M.
A., and Gerwick, W. H. (2004). Structure and biosynthesis of the jamaicamides, new
mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya
majuscula. Chemistry & Biology 11, 817-833.
Gerwick, W. H., Proteau, P. J., Nagle, D., Hamel, E., Blokhin, A., and Slate, D. L.
(1994). Structure of curacin A, a novel antimitotic, antiproliferative, and brine shrimp
toxic natural product from the marine cyanobacterium Lyngbya majuscula. Journal of
Organic Chemistry 59, 1243-1245.
Gerwick,W.H., Tan,L., and Sitachitta,N. (2001). Nitrogen-containing metabolites from
marine cyanobacteria. In The Alkaloids, G.A.Cordell, ed. (San Diego: Academic
Press), pp. 75-184.
Hofflnann, D., Hevel, J. M., Moore, R. E., and Moore, B. S. (2003). Sequence analysis
and biochemical characterization of the nostopeptolide A biosynthetic gene cluster
fromNostocsp. GSV224. Gene 311, 171-180.
Hooper, G. J., Orjala, J., Schatzman, R. C., and Gerwick, W. H. (1998). Carmabins A
and B, new lipopeptides from the Caribbean cyanobacterium Lyngbya majuscula.
Journal of Natural Products 61, 529-533.
Jiménez, J. I. and Scheuer, P. J. (2001). New Lipopeptides from the Caribbean
Cyanobacterium Lyngbya majuscula. Journal of Natural Products 64, 200-203.
151
Julien, B. and Shah, 5. (2002). Heterologous expression of epothilone biosynthetic
genes in Myxococcus xanthus. Antimicrobial Agents and Chemotherapy 46, 27722778.
Malpartida, F. and Hopwood, D. A. (1984). Molecular cloning of the whole
biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous
host. Nature 309, 462-464.
Meeks, J. C., Elhai, J., Thiel, T., Potts, M., Larimer, F., Lamerdin, J., Predki, P., and
Atlas, R. (2001). An overview of the genome of Nostocpunctiforme, a multicellular
symbiotic cyanobacterium. Photosynthesis Research 70, 85-106.
Orjala, J. and Gerwick, W. H. (1996). Barbamide, a chlorinated metabolite with
molluscicidal activity from the Caribbean cyanobacterium Lyngbya majuscula. Journal
of Natural Products 59, 427-430.
Orjala, J., Nagle, D. G., Hsu, V., and Gerwick, W. H. (1995). Antiflatoxin, an
exceptionally ichthyotoxic cyclic lipopeptide from the tropical cyanobacterium
Lyngbya majuscula. Journal of American Chemical Society 117, 828 1-8282.
Recktenwald, J., Shawky, R., Puk, 0., Pfennig, F., Keller, U., Wohileben, W., and
Pelzer, 5. (2002). Nonribosomal biosynthesis of vancomycin-type antibiotics: a
heptapeptide backbone and eight peptide synthetase modules. Microbiology 148,
1105-18.
Schwarzer, D., Finking, R., and Marahiel, M. A. (2003). Nonribosomal peptides: from
genes to products. Natural Product Reports 20, 275-287.
Silakowski, B., Nordsiek, G., Kunze, B., Blocker, H., and Muller, R. (2001). Novel
features in a combined polyketide synthase/non-ribosomal peptide synthetase: the
myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca
Sgal5. Chemistry & Biology 8, 59-69.
Stacheihaus, T. and Marahiel, M. A. (1995). Modular structure of peptide synthetases
revealed by dissection of the multifunctional enzyme GrsA. Journal of Biological
Chemistry 270, 6 163-6169.
Symmank, H., Saenger, W., and Bernhard, F. (1999). Analysis of engineered
multifunctional peptide synthetases - Enzymatic characterization of surfactin
synthetase domains in hybrid bimodular systems. Journal of Biological Chemistry
274, 21581-21588.
Tang, L., Shah, S., Chung, L., Carney, J., Katz, L., Khosla, C., and Julien, B. (2000).
Cloning and heterologous expression of the epothilone gene cluster. Science 287, 640642.
152
Tillett, D., Dittmann, E., Erhard, M., von Dohren, H., Borner, T., and Neilan, B. A.
(2000). Structural organization of micro cystin biosynthesis in Microcystis aeruginosa
PCC7806: an integrated peptide-polyketide synthetase system. Chemistry & Biology
7, 753-764.
Trauger, J. W. and Walsh, C. T. (2000). Heterologous expression in Escherichia coli
of the first module of the nonribosomal peptide synthetase for chioroeremomycin, a
vancomycin-type glycopeptide antibiotic. Proceedings of the National Academy of
Sciences of the United States of America 97, 3112-3117.
Wong, F. C. and Meeks, J. C. (2001). The hetF gene product is essential to heterocyst
differentiation and affects HetR function in the cyanobacterium Nostoc punctiforme.
Journal of Bacteriology 183, 2654-2661.
153
CHAPTER V
CONCLUSIONS
The marine environment is a rich source of biologically active natural products
and continues to be a major source of molecules for drug discovery and development.
Among marine organisms, marine cyanobacteria are proving to be an outstanding
source of biologically active natural products. Moreover, there is growing recognition
that much of the unique chemistry isolated from marine invertebrates is actually of
cyanobacterial origin. The continued discovery of new compounds from these
metabolically talented organisms will provide a range of new chemicals with potential
applications in biotechnology, pharmaceuticals and agriculture. The astounding
structural diversity encountered in cyanobacterial secondary metabolites can be
attributed to their complex biosynthetic systems, consisting of polyketide synthases
and nonribosomal peptide synthetases
This thesis has focused on two secondary metabolite biosynthetic gene clusters
present in a Jamaican isolate of the cyanobacterium Lyngbya
majuscula.
This strain
has been a source of several interesting molecules including hectochiorin, a metabolite
with remarkable antifungal and cytotoxic properties. Initial attempts to clone and
characterize the hectochiorin biosynthetic gene cluster from this organism led to the
identification of a cryptic gene cluster, LMciyl. This allowed us to develop a novel
interdisciplinary approach aimed at identifying the metabolite produced by LMcryl.
The first step in this process was demonstration of its expression by RT-PCR which
was successful. Feeding experiments using labeled amino acid precursors is currently
in progress and identification of the metabolite will be realized using select 2D-NMR
techniques. Genomics guided approaches, such as the one discussed here, will
become a vital part of novel methodologies designed to identify and characterize new
chemical entities. Such methods also allow for the identification of new metabolites
154
present in very low concentrations that may otherwise be missed during routine
chemical isolation procedures.
Using a more specific cloning strategy, the hectochlorin biosynthetic gene
cluster (hct) was subsequently isolated and sequenced. hct adds to the growing
repertoire of marine cyanobacterial gene clusters. The genetic architecture and
domain organization appear to be colinear with respect to its biosynthesis and consists
of eight ORF's spanning 38 kb with several novel features. A striking feature of this
cluster is the contiguity of two ORF's with identical modular organization suggesting
that the cluster may have arisen through a gene duplication event. Another unusual
feature of the cluster is the presence of KR domains in two peptide synthetase modules
which are predicted to be involved in the formation of the two DHiv units and appears
to be the first report of such an arrangement in a cyanobacterial secondary metabolite
gene cluster. Also present are an acyl-ACP synthetase that may be involved in the
activation of a hexanoic acid starter unit, and two cytochrome P450 monooxygenases
which are likely involved in the formation of the DHiv units. A putative halogenase,
at the beginning of the gene cluster, is predicted to form 5,5-dichloro hexanoic acid.
Two adenylation domains from HctE along with the HctA acyl-ACP synthetase were
expressed heterologously in E. coli. While the two adenylation domains were
successfully characterized through biochemical experiments, the acyl-ACP synthetase
awaits further biochemical characterization.
The characterization of each new gene cluster from a marine cyanobacterium
has illustrated the extraordinary versatility of their biosynthetic machinery. Efforts
focused at understanding and dissecting the biosynthesis of these metabolites at the
molecular genetic level will provide the foundation for harnessing these systems
through combinatorial engineering. Additionally, detailed biochemical
characterization of the unique tailoring enzymes associated with cyanobacterial gene
clusters will also facilitate the engineered biosynthesis of novel metabolites. The
development of alternate hosts such a Nostoc punctiforme for the heterologous
expression of individual proteins or entire gene clusters is also of paramount
importance, as such systems can generate an unlimited supply of metabolites for
155
further biomedical or biotechnological use. At present, only a few marine
cyanobacteria are amenable to laboratory cultivation and advances in this area will
enable further investigations of the natural product diversity of these organisms. A
better understanding of the complex metabolic processes governing symbiosis will be
vital in order to exploit the biosynthetic potential of cyanobacterial symbionts and
enhance their application in biotechnology. Finally, whole-genome sequencing of
several cyanobacteria will be crucial for gaining a better understanding of the
regulation of these gene clusters, their arrangement within the genome, their
distribution among different strains and their probable role in the marine environment.
Functional genomic studies which provide a well-defined blueprint of the genome will
be indispensable for the design of rational approaches to search for and harness
secondary metabolite gene clusters involved in the biosynthesis of valuable natural
products.
156
BIBLIOGRAPHY
Aicher, T. D., Buszek, K. R., Fang, F. G., Forsyth, C. J., Jung, S. 0., Kishi, Y.,
Matelich, M. C., Scola, P. M., Spero, D. M., and Yoon, S. K. (1992). Total synthesis
of halichondrin B and norhalichondrin B. Journal of American Chemical Society 114,
3 162-3 164.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic
local alignment search tool. Journal of Molecular Biology 215, 403-410.
Amador, M. L., Jimeno J., Paz-Ares L., Cortes-Funes H., and Hidago M. (2003).
Progress in the developement and acquisition of anticancer agents from marine
sources. Annals of Oncology 14, 1607-1615.
Andersen, J. F. and Hutchinson, C. R. (1992). Characterization of Saccharopolyspora
erythraea cytochrome P-450 genes and enzymes, including 6-deoxyerythronolide B
hydroxylase. Journal of Bacteriology 174, 725-735.
Bancroft, I. and Wolk, C. P. (1989). Characterization of an insertion sequence (1S891)
of novel structure from the cyanobacterium Anabaena sp. strain M-1 31. Journal of
Bacteriology 171, 5949-5954.
Bai, R., Pettit, G. R., and Hamel, B. (1990). Dolastatin 10, a powerful cytostatic
peptide derived from a marine animal. Inhibition of tubulin polymerization mediated
through the ymca alkaloid binding domain. Biochemical Pharmacology 39, 194 11949.
Barry,B.A., Boerner,R.J., and de Paula,J.C. (1994). The use of cyanobacteria in the
study of the structure and function of photosystem II. In The molecular biology of
cyanobacteria, D.A.Bryant, ed. (Netherlands: Kiuwer Academic Publishers), pp. 217257.
Batard, P., Szollosi, J., Luescher, I., Cerottini, J. C., MacDonald, R., and Romero, P.
(2002). Use of phycoerythrin and allophycocyanin for fluorescence resonance energy
transfer analyzed by flow cytometry: advantages and limitations. Cytometry 48, 97105.
Becker, J. E., Moore, R. E., and Moore, B. 5. (2004). Cloning,sequencing,and
biochemical characterization of the nostocyclopeptide biosynthetic gene
cluster:molecular basis for imine macrocyclization. Gene 325, 35-42.
Belshaw, P. J., Walsh, C. T., and Stachelhaus, T. (1999). Aminoacyl-CoAs as probes
of condensation domain selectivity in nonribosomal peptide synthesis. Science 284,
486-489.
157
Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M., Challis, G. L., Thomson, N. R.,
James, K. D., Harris, D. E., Quail, M. A., Kieser, H., Harper, D., Bateman, A., Brown,
S., Chandra, G., Chen, C. W., Collins, M., Cronin, A., Fraser, A., Goble, A., Hidalgo,
J., Homsby, T., Howarth, S., Huang, C. H., Kieser, T., Larke, L., Murphy, L., Oliver,
K., O'Neil, S., Rabbinowitsch, E., Rajandream, M. A., Rutherford, K., Rutter, S.,
Seeger, K., Saunders, D., Sharp, S., Squares, R., Squares, S., Taylor, K., Warren, T.,
Wietzorrek, A., Woodward, J., Barrell, B. G., Parkhill, J., and Hopwood, D. A. (2002).
Complete genome sequence of the model actinomycete Streptomyces coelicolor
A3(2). Nature 417, 141-7.
Bergman, W. and Feeney, R. J. (1951). Marine products. XXXII. The nucelosides of
sponges. I. Journal of Organic Chemistry 16, 981-987.
Berman, F. W., Gerwick, W. H., and Murray, T. F. (1999). Antillatoxin and
kalkitoxin, ichtbyotoxins from the tropical cyanobacterium Lyngbya majuscula,
induce distinct temporal patterns of NMDA receptor-mediated neurotoxicity. Toxicon
37, 1645-8.
Beyer, S., Kunze, B., Silakowski, B., and Muller, R. (1999). Metabolic diversity in
myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene
cluster of Stigmatella aurantiaca Sg a15 and a combined polyketide-Qoly)peptide
gene cluster from the epothilone producing strain Sorangium cellulosum So ce9O.
Biochimica Et Biophysica Acta 1445, 185-195.
Biemian, M., Logan, R., O'Brien, K., Seno, E. T., Rao, R. N., and Schoner, B. E.
(1992). Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia
coli to Streptomyces spp. Gene 116, 43-49.
Blunt, J. W., Copp, B. R., Munro, M. H., Northcote, P. T., and Prinsep, M. R. (2005).
Marine natural products. Natural Product Reports 22, 15-61.
Boddy, C. N., Hotta, K., Tse, M. L., Watts, R. E., and Khosla, C. (2004). Precursordirected biosynthesis of epothilone in Escherichia coli. Journal of American Chemical
Society 126, 7436-7437.
Boddy, C. N., Schneider, T. L., Hotta, K., Walsh, C. T., and Khosla, C. (2003).
Epothilone C macrolactonization and hydrolysis are catalyzed by the isolated
thioesterase domain of epothilone polyketide synthase. Journal of American Chemical
Society 125, 3428-3429.
Briand, J. F., Jacquet, S., Bernard, C., and Humbert, J. F. (2003). Health hazards for
terrestrial vertebrates from toxic cyanobacteria in surface water ecosystems.
Veterinary Research 34, 361-377.
158
Burgoyne, D. L., Andersen, R. J., and Allen, T. M. (1992). Contignasterol, a highly
oxygenated steroid with the unnatural 14b configuration from the marine sponge
Petrosia contignata Thiele, 1899. Journal of Organic Chemistry 57, 525-528.
Burja, A. M., Banaigs, B., Abou-Mansour, E., Burgess, J. G., and Wright, P. C.
(2001). Marine cyanobacteria: A prolific source of natural products. Tetrahedron 57,
9347-9377.
Cane, D. E. and Walsh, C. T. (1999). The parallel and convergent universes of
polyketide synthases and nonribosomal peptide synthetases. Chemistry & Biology 6,
3 19-325.
Cane, D. B., Walsh, C. T., and Khosla, C. (1998). Harnessing the biosynthetic code:
combinations, permutations, and mutations. Science 282, 63-68.
Cardellina, J. H., Marner, F. J., and Moore, R. E. (1979). Seaweed dermatitis: structure
of lyngbyatoxin A. Science 204, 193-195.
Carmichael, W. W., Eschedor, J. T., Patterson, G. M., and Moore, R. E. (1988).
Toxicity and partial structure of a hepatotoxic peptide produced by the
cyanobacterium Nodularia spumigena Mertens emend. L575 from New Zealand.
Applied and Environmental Microbiology 54, 2257-2263.
Castenholz,R.W., Rippka,R., and Herdman,M. (2001). Bergey's Manual of Systematic
Bacteriology Volume I. (New York: Springer-Verlag).
Censini, S., Lange, C., Xiang, Z., Crabtree, J. E., Ghiara, P., Borodovsky, M.,
Rappuoli, R., and Covacci, A. (1996). cag, a pathogenicity island of Helicobacter
pylon, encodes type I-specific and disease-associated virulence factors. Proceedings of
the National Academy of Sciences of the United States of America 93, 14648-14653.
Cetusic, J. R. P., Green, F. R., Graupner, P. R., and Oliver, M. P. (2002). Total
Synthesis of Hectochlorin. Organic Letters 4, 1307-13 10.
Challis, G. L., Ravel, J., and Townsend, C. A. (2000). Predictive, structure-based
model of amino acid recognition by nonribosomal peptide synthetase adenylation
domains. Chemistry & Biology 7, 211-224.
Challis, G. L. and Ravel, J. (2000). Coelichelin, a new peptide siderophore encoded by
the Streptomyces coelicolor genome: structure prediction from the sequence of its nonribosomal peptide synthetase. FEMS Microbiology Letters 187, 111-114.
Chang, Z., Flatt, P., Gerwick, W. H., Nguyen, V. A., Willis, C. L., and Sherman, D. H.
(2002). The barbamide biosynthetic gene cluster: a novel marine cyanobacterial
system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS)
origin involving an unusual trichioroleucyl starter unit. Gene 296, 235-247.
159
Chang, Z., Sitachitta, N,, Rossi, J. V., Roberts, M. A., Flatt, P. M., Jia, J., Sherman, D.
H., and Gerwick, W. H. (2004). Biosynthetic pathway and gene cluster analysis of
curacin A, an antitubulin natural product from the tropical marine cyanobacterium
Lyngbya majuscula. Journal of Natural Products 67, 1356-1367.
Chen, H., Hubbard, B. K., O'Connor, S. E., and Walsh, C. T. (2002). Formation of
beta-hydroxy histidine in the biosynthesis of nikkomycin antibiotics. Chemistry &
Biology 9, 103-112.
Chen, H., Thomas, M. G., O'Connor, S. E., Hubbard, B. K., Burkart, M. D., and
Walsh, C. T. (2001). Aminoacyl-S-enzyme intermediates in beta-hydroxylations and
alpha,beta- desaturations of amino acids in peptide antibiotics. Biochemistry 40,
1165 1-9.
Chen, H. and Walsh, C. T. (2001). Coumarin formation in novobiocin biosynthesis:
beta-hydroxylation of the aminoacyl enzynle tyrosyl-S-NovH by a cytochrome P450
NovI. Chemistry & Biology 8, 301-3 12.
Chen, J. and Forsyth, C. J. (2004). Total synthesis of the marine cyanobacterial
cyclodepsipeptide apratoxin A. Proc Nati Acad Sci U S A 101, 12067-12072.
Christiansen, G., Fastner, J., Erhard, M., Borner, T., and Dittmann, E. (2003).
Microcystin biosynthesis in Planktothrix: genes, evolution, and manipulation. J
Bacteriol 185, 564-72.
Conti, E., Stacheihaus, T., Marahiel, M. A., and Brick, P. (1997). Structural basis for
the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S.
Embo Journal 16,4174-4183.
Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J., and Leadlay, P. F. (1990). An
unusually large multifunctional polypeptide in the erythromycin- producing polyketide
synthase of Saccharopolyspora erythraea. Nature 348, 176-8.
Coulson, F. R. and O'Donnell, S. R. (2000). The effects of contignasterol (IZP-94,005)
on allergen-induced plasma protein exudation in the tracheobronchial airways of
sensitized guinea-pigs in vivo. Inflammation Research 49, 123-127.
Donadio, S., Stayer, M. J., McAlpine, J. B., Swanson, S. J., and Katz, L. (1991).
Modular organization of genes required for complex polyketide biosynthesis. Science
252, 675-679.
Donadio, S. and Stayer, M. J. (1993). 1S1136, an Insertion Element in the
Erythromycin Gene Cluster of Saccharopolyspora erythraea. Gene 126, 147-15 1.
Donia, M. and Hamann, M. T. (2003). Marine natural products and their potential
applications as anti-infective agents. Lancet Infectious Diseases 3, 338-348.
160
Dougherty, T. J., Barrett, J. F., and Pucci, M. J. (2002). Microbial genomics and novel
antibiotic discovery: new technology to search for new drugs. Current Pharmaceutical
Design 8, 1119-1135.
Du, L., Sanchez, C., Chen, M., Edwards, D. J., and Shen, B. (2000). The biosynthetic
gene cluster for the antitumor drug bleomycin from Streptomyces verticillus
ATCC 15003 supporting functional interactions between nonribosomal peptide
synthetases and a polyketide synthase. Chemistry & Biology 7, 623-642.
Du, L., Chen, M., Sanchez, C., and Shen, B. (2000). An oxidation domain in the
Bimill non-ribosomal peptide synthetase probably catalyzing thiazole formation in the
biosynthesis of the anti- tumor drug bleomycin in Streptomyces verticillus
ATCC15003. FEMS Microbiology Letters 189, 171-175.
Edwards, D. J. and Gerwick, W. H. (2004). Lyngbyatoxin biosynthesis: sequence of
biosynthetic gene cluster and identification of a novel aromatic prenyltransferase.
Journal of American Chemical Society 126, 11432-11433.
Edwards, D. J., Marquez, B. L., Nogle, L. M., McPhail, K., Goeger, D. E., Roberts, M.
A., and Gerwick, W. H. (2004). Structure and biosynthesis ofthejamaicamides, new
mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya
majuscula. Chemistry & Biology 11, 8 17-833.
Egner, P. A., Wang, J. B., Zhu, Y. R., Zhang, B. C., Wu, Y., Zhang, Q. N., Qian, G.
S., Kuang, S. Y., Gange, S. J., Jacobson, L. P., Helzlsouer, K. J., Bailey, G. S.,
Groopman, J. D., and Kensler, T. W. (2001). Chlorophyllin intervention reduces
aflatoxin-DNA adducts in individuals at high risk for liver cancer. Proceedings of the
National Academy of Sciences of the United States of America 98, 14601-14606.
Ehling-Schulz, M., Vukov, N., Schulz, A., Shaheen, R., Andersson, M., Martlbauer,
E., and Scherer, 5. (2005). Identification and partial characterization of the
nonribosomal peptide synthetase gene responsible for cereulide production in emetic
Bacillus cereus. Applied and Environmental Microbiology 71, 105-113.
Ehmann, D. E., Trauger, J. W., Stachelhaus, T., and Walsh, C. T. (2000). AminoacylSNACs as small-molecule substrates for the condensation domains of nonribosomal
peptide synthetases. Chemistry & Biology 7, 765-772.
Erba, E., Bergamaschi, D., Ronzoni, S., Faretta, M., Tavema, S., Bonfanti, M.,
Catapano, C. V., Faircloth, G., Jimeno, J., and D'Inalci, M. (1999). Mode of action of
thiocoraline, a natural marine compound with antitumor activity. Bristish Journal of
Cancer 80, 1901-1907.
Fehling, R. H., Buchanan, G. 0., Mincer, T. J., Kauffman, C. A., Jensen, P. R., and
Fenical, W. (2003). Salinosporamide A: a highly cytotoxic proteasome inhibitor from
161
a novel microbial source, a marine bacterium of the new genus Salinospora. Angew
Chem Tnt Ed Engi 42, 355-357.
Fenteany, G. and Zhu, S. (2003). Small-molecule inhibitors of actin dynamics and cell
motility. Current Topics in Medicinal Chemistry 3, 593-6 16.
Ferber, M., Sporning, A., Jeserich, G., DeLaCruz, R., Watkins, M., Olivera, B. M.,
and Terlau, H. (2003). A novel conus peptide ligand for K+ channels. Journal of
Biological Chemistry 278, 2177-2183.
Finking, R. and Marahiel, M. (2004). Biosynthesis of nonribosomal peptides. Annual
Review of Microbiology 58, 453-488.
Fiore, M. F., Moon, D. H., Tsai, S. M., Hung, L., and Trevors, J. T. (2000). Miniprep
DNA isolation from unicellular and filamentous cyanobacteria. Journal of
Microbiological Methods 39, 159-169.
Fujii,K., Harada,K.-I., Suzuki,M., Kondo,F., Ikai,Y., Oka,H., and et al. (1996).
Occurence of novel cyclic depsipeptides togteher with microcystins from toxic
cyanobacteria Anabaena species. In Harmful and Toxic Algal Blooms, T.Yasumoto,
Y.Oshima, and Y.Fukuyo, eds. (Paris: Intergovernmental Oceanographic Commission
of UNESCO), pp. 5 59-562.
Fujiki, H., Suganuma, M., Hakii, H., Bartolini, G., Moore, R. E., Takayama, S., and
Sugimura, T. (1984). A two-stage mouse skin carcinogenesis study of lyngbyatoxin A.
Journal of Cancer Research and Clinical Oncology 108, 174-6.
Gaidenko, T. A. and Khaikinson, M. (1988). Cloning of the genes controlling the
biosynthesis of bacitracin in Bacillus lichenformis. Molekuliarnaia genetika,
mikrobiologiia i virusologiia 2, 24-28.
Garcia-Pichel, F. and Castenholz, R. (1991). Characterization and biological
implications of scytonemin, a cyanobacterial sheath pigment. Journal of Phycology 27,
395-405.
Garcia-Pichel, F., Sherry, N. D., and Castenholz, R. (1992). Evidence for an
ultraviolet sunscreen role of the extracellular pigment scytonemin in the terrestrial
cyanobacterium, Chiorogleopsis sp. Photochemistry and Photobiology 56, 17-23.
Garcia-Rocha, M., Bonay, P., and Avila, J. (1996). The antitumoral compound
kahalalide F acts on cell lysosomes. Cancer Letters 99, 43-50.
Gehring, A. M., Mori, I., Perry, R. D., and Walsh, C. T. (1998). The nonribosomal
peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of
yersiniabactin, an iron-chelating virulence factor of Yersinia pestis. Biochemistry 37,
11637-11650.
162
Gerwick, W. H., Roberts, M. A., Proteau, P. J., and Chen, J.-L. (1994). Screening
cultured marine microalgae for anticancer-type activity. Journal of Applied Phycology
6, 143-149.
Gerwick, W. H., Proteau, P. J., Nagle, D., Hamel, E., Blokhin, A., and Slate, D. L.
(1994). Structure of curacin A, a novel antimitotic, antiproliferative, and brine shrimp
toxic natural product from the marine cyanobacterium Lyngbya majuscula. Journal of
Organic Chemistry 59, 1243-1245.
Gerwick,W.H., Tan,L., and Sitachitta,N. (2001). Nitrogen-containing metabolites from
marine cyanobacteria. In The Alkaloids, G.A.Cordell, ed. (San Diego: Academic
Press), pp. 75-184.
Giovannoni, S. J., Turner, S., Olsen, G. J., Barns, S., Lane, D. J., and Pace, N. R.
(1988). Evolutionary relationships among cyanobacteria and green chioroplasts.
Journal of Bacteriology 170, 3584-3592.
Golakoti, T., Ohtani, I., Patterson, G. M., Moore, R. E., Corbett, T. H., Valeriote, F.
A., and Demchik, L. (1994). Total Structures of Cryptophycins, Potent Antitumor
Depsipeptides from the Blue-Green Alga Nostoc sp. Strain GSV 224. Journal of
American Chemical Society 116, 4729-4737.
Grassle, J. F. and Maciolek, N. J. (1992). Deep-sea species richness: regional and local
diversity estimates from quantitative bottom samples. American Naturalist 139, 313341.
Gruenewald, S., Mootz, H. D., Stehmeier, P., and Stachelhaus, T. (2004). In vivo
production of artificial nonribosomal peptide products in the heterologous host
Escherichia coli. Applied and Environmental Microbiology 70, 3282-329 1.
Guenzi, E., Galli, G., Grgurina, I., Gross, D. C., and Grandi, G. (1998).
Characterization of the syringomycin synthetase gene cluster A link between
prokaryotic and eukaryotic peptide synthetases. Journal of Biological Chemistry 273,
32857-32863.
Gunasekera, S. P., Gunasekera, M., Longley, R. E., and Schulte, G. K. (1990).
Discodermolide: a new bioactive polyhydroxylated lactone from the marine sponge
Discodermia dissoluta. Journal of Organic Chemistry 55, 49 12-4915.
Hahn, M. and Stacheihaus, T. (2004). Selective interaction between nonribosomal
peptide synthetases is facilitated by short communication-mediating domains.
Proceedings of the National Academy of Sciences of the United States of America
101, 15585-15590.
163
Hamann, M. T. and Scheuer, P. J. (1993). Kahalalide F: a bioactive depsipeptide from
the sacoglossan mollusk Elysia rufescens and the green alga Bryopsis sp. Journal of
American Chemical Society 115, 5825-5826.
Hanson, R. L., Singh, J., Kissick, T. P., Patel, R. N., Szarka, L. J., and Mueller, R. H.
(1990). Synthesis of L-3Hydroxyvaline from aKeto-hydroxy isovalerate using
leucine dehydrogenase from Bacillus sp. Bioorganic Chemistry 18, 116-130.
Haydock, S. F., Aparicio, J. F., Molnar, I., Schwecke, T., Khaw, L. E., Konig, A.,
Marsden, A. F., Galloway, I. S., Staunton, J., and Leadlay, P. F. (1995). Divergent
sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl
carrier protein transacylase domains in modular polyketide synthases. Febs Letters
374, 246-248.
Hoffmnann, D., Hevel, J. M., Moore, R. B., and Moore, B. S. (2003). Sequence analysis
and biochemical characterization of the nostopeptolide A biosynthetic gene cluster
from Nostoc sp. GSV224. Gene 311, 171-180.
Hooper, G. J., Orjala, J., Schatzman, R. C., and Gerwick, W. H. (1998). Carmabins A
and B, new lipopeptides from the Caribbean cyanobacterium Lyngbya majuscula.
Journal of Natural Products 61, 529-533.
Hori, K., Yamomoto, Y., Minetoki, T., Kurotsu, T., Kanda, M., Miura, S., Okamura,
F., Furuyama, J., and Saito, Y. (1989). Molecular cloning and nucleotide sequence of
the gramicidin S synthetase 1 gene. Journal of Biochemistry (Tokyo) 106, 639-645.
Ishida, T., Watanabe, M. M., Sugiyama, J., and Yokota, A. (2001). Evidence for
polyphyletic origin of the members of the orders of Oscillatoriales and Pleurocapsales
as determined by their 16S rDNA analysis. FEMS Microbiology Letters 201, 79-82.
Iwasaki, H. and Kondo, T. (2004). Circadian timing mechanism in the prokaryotic
clock system of cyanobacteria. Journal of Biological Rhythms 19, 348-360.
Jensen, P. R., Mincer, T. J., Williams, P. G., and Fenical, W. (2005). Marine
actinomycete diversity and natural product discovery. Antonie Van Leeuwenhoek 87,
43-48.
Jiménez, J. I. and Scheuer, P. J. (2001). New Lipopeptides from the Caribbean
Cyanobacterium Lyngbya majuscula. Journal of Natural Products 64, 200-203.
Jimeno, J., Lopez-Martin, J. A., Ruiz-Casado, A., Izquierdo, M. A., Scheuer, P. J., and
Rinehart, K. (2004). Progress in the clinical development of new marine-derived
anticancer compounds. Anticancer Drugs 15, 32 1-329.
164
Julien, B. and Shah, S. (2002). Heterologous expression of epothilone biosynthetic
genes in Myxococcus xanthus. Antimicrobial Agents and Chemotherapy 46, 27722778.
Kaebernick, M., Neilan, B. A., Borner, T., and Dittmann, B. (2000). Light and the
transcriptional response of the microcystin biosynthesis gene cluster [In Process
Citation]. Applied and Environmental Microbiology 66, 33 87-92.
Kaebernick, M., Dittmann, B., Borner, T., and Neilan, B. A. (2002). Multiple alternate
transcripts direct the biosynthesis of microcystin, a cyanobacterial nonribosomal
peptide. Applied and Environmental Microbiology 68, 449-55.
Kalesse, M. (2000). The chemistry and biology of discodermolides. Chembiochem 1,
171-175.
Kaneko, T., Nakamura, Y., Wolk, C. P., Kuritz, T., Sasamoto, S., Watanabe, A.,
Iriguchi, M., Ishikawa, A., Kawashima, K., Kimura, T., Kishida, Y., Kohara, M.,
Matsumoto, M., Matsuno, A., Muraki, A., Nakazaki, N., Shimpo, S., Sugimoto, M.,
Takazawa, M., Yamada, M., Yasuda, M., and Tabata, S. (2001). Complete genomic
sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC
7120. DNA Research 8, 205-213.
Katz, L. (1997). Manipulation of modular polyketide synthases. Chemical Reviews 97,
2557-2575.
Kennell, D. (2002). Processing endoribonulceases and mRNA degradation in bacteria.
Journal of Bacteriology 184, 4645-4657.
Kerksiek, K., Mejillano, M. R., Schwartz, R. B., Georg, G. I., and Himes, R. H.
(1995). Interaction of cryptophycin 1 with tubulin and microtubules. Febs Letters 377,
59-61.
Kershaw, N. J., Mukherji, M., MacKinnon, C. H., Claridge, T. D., Odell, B.,
Wierzbicki, A. S., Lloyd, M. D., and Schofield, C. J. (2001). Studies on phytanoylCoA 2-hydroxylase and synthesis of phytanoyl-coenzyme A. Bioorganic & Medicinal
Chemistry 11, 2545-2548.
Khosla, C. (1997). Harnessing the biosynthetic potential of modular polyketide
synthases. Chemical Reviews 97, 2577-2590.
Kobayashi, M., Aoki, S., Ohyabu, N., Kurosum, M., Wang, W., and Kitagawa, I.
(1994). Arenastatin A, a potent cytotoxic depsipeptide from the Okinawan marine
sponge Dysidea arenaria. Tetrahedron Letters 35, 7969-7972.
165
Kobayashi, M., Natsume, T., Tamaoki, S., Watanabe, J., Asano, H., Mikami, T.,
Miyasaka, K., Miyazaki, K., and Gondo, M. (1997). Antitumor activity of TZT-1027,
a novel dolastatin 10 derivative. Japanese Journal of Cancer Research 88, 316-327.
Kohli, R. M., Takagi, J., and Walsh, C. T. (2002a). The thioesterase domain from a
nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides.
Proceedings of the National Academy of Sciences of the United States of America 99,
1247-1252.
Kohli, R. M., Trauger, J. W., Schwarzer, D., Marahiel, M. A., and Walsh, C. T.
(2001). Generality of peptide cyclization catalyzed by isolated thioesterase domains of
nonribosomal peptide synthetases. Biochemistry 40, 7099-7 108.
Kohli, R. M. and Walsh, C. T. (2003). Enzymology of acyl chain macrocyclization in
natural product biosynthesis. Chemical Communication (Camb) 7, 297-307.
Kohli, R. M., Walsh, C. T., and Burkart, M. D. (2002b). Biomimetic synthesis and
optimization of cyclic peptide antibiotics. Nature 418, 658-661.
Konz, D., Kiens, A., Schorgendorfer, K., and Marahiel, M. A. (1997). The bacitracin
biosynthesis operon of Bacillus lichenformis ATCC 10716: molecular
characterization of three multi-modular peptide synthetases. Chemistry & Biology 4,
927-937.
Kratzschmar, J., Krause, M., and Marahiel, M. A. (1989). Gramicidin-S Biosynthesis
Operon Containing the Structural Genes Grsa and Grsb Has an Open Reading Frame
Encoding a Protein Homologous to Fatty Acid Thioesterases. Journal of Bacteriology
171, 5422-5429.
Krishnamurthy, T., Carmichael, W. W., and Sarver, E. W. (1986). Toxic peptides from
freshwater cyanobacteria (blue-green algae). I. Isolation, purification and
characterization of peptides from Microcystis aeruginosa and Anabaena flos-aquae.
Toxicon 24, 865-873.
Kuse, M., Franz, T., Koga, K., Suwan, S., Isobe, M., Agata, N., and Ohta, M. (2000).
High incorporation of L-amino acids to cereulide, an emetic toxin from Bacillus
cereus. Bioorganic & Medicinal Chemistry Letters 10, 735-739.
Kushner, S. R. (2002). mRNA decay in Escherichia coli comes of age. Journal of
Bacteriology 184, 4658-4665.
Li, W. I., Berman, F. W., Okino, T., Yokokawa, F., Shioiri, T., Gerwick, W. H., and
Murray, T. F. (2001). Antillatoxin is a marine cyanobacterial toxin that potently
activates voltage-gated sodium channels. Proceedings of the National Academy of
Sciences of the United States of America 98, 7599-7604.
166
Lindel, T., Jensen, P. R., Fenical, W., Long, B. H., Casazza, A.-M., Carboni, J., and
Fairchild, C. R. (1997). Eleutherobin, a New Cytotoxin that Mimics Paclitaxel (Taxol)
by Stabilizing Microtubules. Journal of American Chemical Society 119, 8744-8745.
Liu, J., Duncan, K., and Walsh, C. T. (1989). Nucleotide sequence of a cluster of
Escherichia coli enterobactin biosynthesis genes: identification of entA and
purification of its product 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase. Journal
of Bacteriology 171, 791-798.
Long, B. H., Carboni, J. M., Wasserman, A. J., Cornell, L. A., Casazza, A.-M., Jensen,
P. R., Lindel, T., Fenical, W., and Fairchild, C. R. (1998). Eleutherobin, a novel
cytotoxic agent that induces tubulin polymerization is similar to paclitaxel (Taxol).
Cancer Research 58, 1111-1115.
Luesch, H., Moore, R. E., Paul, V. J., Mooberry, S. L., and Corbett, T. H. (2001a).
Isolation of dolastatin 10 from the marine cyanobacterium Symploca species VP642
and total stereochemistry and biological evaluation of its analogue symplostatin 1.
Journal of Natural Products 64, 907-910.
Luesch, H., Yoshida, W. Y., Moore, R. E., and Paul, V. J. (2000a). Isolation and
structure of the cytotoxin lyngbyabellin B and absolute configuration of lyngbyapeptin
A from the marine cyanobacterium Lyngbya majuscula. Journal of Natural Products
63, 1437-1439.
Luesch, H., Yoshida, W. Y., Moore, R. E., Paul, V. J., and Corbett, T. H. (2001b).
Total structure determination of apratoxin A, a potent novel cytotoxin from the marine
cyanobacterium Lyngbya majuscula. Journal of American Chemical Society 123,
5418-5423.
Luesch, H., Yoshida, W. Y., Moore, R. E., Paul, V. J., and Mooberry, S. L. (2000b).
Isolation, structure determination, and biological activity of Lyngbyabellin A from the
marine cyanobacterium Lyngbya majuscula. Journal of Natural Products 63, 611-615.
Maccabe, A. P., Vanliempt, H., Palissa, H., Unkles, S. E., Riach, M. B. R., Pfeifer, E.,
vonDohren, H., and Kinghorn, J. R. (1991). Delta-(L-Alpha-Aminoadipyl)-LCysteinyl-D-Valine Synthetase from Aspergillus-Nidulans - Molecular
Characterization of the Acva Gene Encoding the 1st Enzyme of the Penicillin
Biosynthetic Pathway. Journal of Biological Chemistry 266, 12646-12654.
MacNeil, D. J., Occi, J. L., Gewain, K. M., MacNeil, T., Gibbons, P. H., Ruby, C. L.,
and Danis, S. J. (1992). Complex organization of the Streptomyces avermitilis genes
encoding the avermectin polyketide synthase. Gene 115, 119-125.
Mahillon, J., Leonard, C., and Chandler, M. (1999). IS elements as constituents of
bacterial genomes. Research in Microbiology 150, 675-687.
167
Mahillon, J. and Chandler, M. (1998). Insertion sequences. Microbiology and
Molecular Biology Reviews 62, 725-774.
Malpartida, F. and Hopwood, D. A. (1984). Molecular cloning of the whole
biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous
host. Nature 309, 462-464.
Manzanares, I., Cueves, C., Garcia-Nieto, R., Marco, E., and Gago, F. (2001).
Advances in the chemistry and pharmacology of ecteinascidins, a promising new class
of anti-cancer agents. Current Medicinal Chemistry and Anticancer Agents 1, 257276.
Marahiel, M. A., Stacheihaus, T., and Mootz, H. D. (1997). Modular peptide
synthetases involved in nonribosomal peptide synthesis. Chemical Reviews 97, 265 12673.
Marquez, B. L., Watts, K. S., Yokochi, A., Roberts, M. A., Verdier-Pinard, P.,
Jimenez, J. I., Hamel, E., Scheuer, P. J., and Gerwick, W. H. (2002). Structure and
absolute stereochemistry of hectochiorin, a potent stimulator of actin assembly.
Journal of Natural Products 65, 866-871.
May, R. M. (2005). Biological diversity: differences between land and sea.
Philosophical Transactions of the Royal Society of London Series B-Biological
Sciences 343, 105-111.
McAlpine, J. B., Bachmaim, B. 0., Piraee, M., Tremblay, S., Alarco, A.-M.,
Zazopoulos, E., and Farnet, C. M. (2005). Mircobial genomics as a guide to drug
discovery and structural elucidation: ECO-0230 1, a novel antifungal agent, as an
example. Journal of Natural Products 68, 493-496.
McDaniel, R., Welch, M., and Hutchinson, C. R. (2005). Genetic approaches to
polyketide antibiotics. 1. Chemical Reviews 105, 543-558.
Meeks, J. C., Elhai, J., Thiel, T., Potts, M., Larimer, F., Lamerdin, J., Predki, P., and
Atlas, R. (2001). An overview of the genorne of Nostocpunctforme, a multicellular
symbiotic cyanobacterium. Photosynthesis Research 70, 85-106.
Meeks, J. C., Elhai, J., Thiel, T., Potts, M., Larimer, F., Lamerdin, J., Predki, P., and
Atlas, R. (2001). An overview of the genome of Nostoc punctforme, a multicellular
symbiotic cyanobacterium. Photosynthesis Research 70, 85-106.
Milligan, K. E., Marquez, B. L., Williamson, R. T., and Gerwick, W. H. (2000).
Lyngbyabellin B, a toxic and antifungal secondary metabolite from the marine
cyanobacterium Lyngbya majuscula. Journal of Natural Products 63, 1440-3.
Miyazaki, K., Kobayashi, M., Natsume, T., Gondo, M., Mikami, T., Sakakibara, K.,
and Tsukagoshi, S. (1995). Synthesis and antitumor activity of novel dolastatin 10
analogs. Chemical & pharmaceutical bulletin 43, 1706-1718.
Moffitt, M. C. and Neilan, B. A. (2004). Characterization of the nodularin synthetase
gene cluster and proposed theory of the evolution of cyanobacterial hepatotoxins.
Applied and Environmental Microbiology 70, 6353-6362.
Molnar, I., Schupp, T., Ono, M., Zirkie, R., Milnamow, M., Nowak-Thompson, B.,
Engel, N., Toupet, C., Stratmann, A., Cyr, D. D., Gorlach, J., Mayo, J. M., Hu, A.,
Goff, S., Schmid, J., and Ligon, J. M. (2000). The biosynthetic gene cluster for the
microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So
ce9O. Chemistry & Biology 7, 97-109.
Moore, B. S., Hertweck, C., Hopke, J. N., Izumikawa, M., Kalaitzis, J. A., Nilsen, G.,
O'Hare, T., Piel, J., Shipley, P. R., Xiang, L., Austin, M. B., and Noel, J. P. (2002).
Plant-like Biosynthetic Pathways in Bacteria: From Benzoic Acid to Chalcone(1).
Journal of Natural Products 65, 1956-1962.
Moore, B. S. and Hopke, J. (2001). Discovery of new bacterial polyketide biosynthetic
pathway. Chembiochem 2, 35-38.
Moore,R.E. (1978). Algal nonisoprenoids. In Marine Natural Products, Chemical and
Biological Perspectives, P.J.Scheuer, ed. (New York: Academic Press), pp. 43-124.
Moore,R.E. (1981). Constituents of the blue-green alga. In Marine Natural
Products: Chemical and Biological Perspectives, P.J. Scheuer, ed. (London: Academic
Press), pp. 1-52.
Moore, R. E. (1996). Cyclic peptides and depsipeptides from cyanobacteria: a review.
Journal of Industrial Microbiology 16, 134-143.
Moore,R.E., Patterson,G.M.L., and Carmichael,W.W. (1988). New pharmaceuticals
from cultured blue-green algae. In Biomedical Importance of Marine Organisms,
D.G.Fautin, ed. (San Francisco: California Academy of Sciences), pp. 143-150.
Motamedi, H., Cai, S. J., Shaflee, A., and Elliston, K. 0. (1997). Structural
organization of a multifunctional polyketide synthase involved in the biosynthesis of
the macrolide immunosuppressant FK506. European Journal of Biochemistry 244, 74
80.
Mukherji, M., Chien, W., Kershaw, N. J., Clifton, I. J., Schofield, C. J., Wierzbicki, A.
S., and Lloyd, M. D. (2001). Structure-function analysis of phytanoyl-CoA 2hydroxylase mutations causing Refsum's disease. Human Molecular Genetics 10,
1971-1982.
Murai, N., Kamata, H., Nagashima, Y., Yagisawa, H., and Hirata, H. (1995). A novel
insertion sequence (IS)-like element of the thermophilic bacterium PS3 promotes
expression of the alanine carrier protein-encoding gene. Gene 163, 103-107.
Mynderse, J. S., Moore, R. E., Kashiwagi, M., and Norton, T. R. (1977). Antileukemia
activity in the Oscillatoriaceae: isolation of debromoaplysiatoxin from Lyngbya.
Science 196, 538-540.
Nageli, C. (1849). Neue Denkschr. Aug. Schwiezer Ges. Naturwissenschaften 10, 1.
Nagle, D. G. and Paul, V. J. (1999). Production of secondary metabolites by
filamentous tropical marine cyanobacteria:ecological functions of the compounds.
Journal of Phycology 35, 1412-1421.
Nagle, D. G. and Paul, V. J. (1998). Chemical defense of a marine cyanobacterial
bloom. Journal of Experimental Marine Biology and Ecology 225, 29-3 8.
Neilan, B. A., Dittmann, E., Rouhiainen, L., Bass, R. A., Schaub, V., Sivonen, K., and
Borner, T. (1999). Nonribosomal peptide synthesis and toxigenicity of cyanobacteria.
Journal of Bacteriology 181, 4089-4097.
Newman, D. J. and Cragg, G. M. (2004b). Marine Natural Products and Related
Compounds in Clinical and Advanced Preclinical Trials. Journal of Natural Products
67, 1216-1238.
Newman, D. J. and Cragg, G. M. (2004a). Advanced preclinical and clinical trials of
natural products and related compounds from marine sources. Current Medicinal
Chemistry 11, 1693-1713.
Newman, D. J., Cragg, G. M., and Snader, K. M. (2003). Natural products as sources
of new drugs over the period 1981-2002. Journal of Natural Products 66, 1022-1037.
Newman, D. J., Cragg, G. M., and Snader, K. M. (2000). The influence of natural
products upon drug discovery. Natural Product Reports 17, 215-234.
Nogle, L. M. Structure elucidation and biosynthetic investigations of marine
cyanobacterial secondary metabolites. 2002. Oregon State University.
Ref Type: Thesis/Dissertation
Omura, S., Ikeda, H., Ishikawa, J., Hanamoto, A., Takahashi, C., Shinose, M.,
Takahashi, Y., Horikawa, H., Nakazawa, H., Osonoe, T., Kikuchi, H., Shiba, T.,
Sakaki, Y., and Hattori, M. (2001). Genome sequence of an industrial microorganism
Streptomyces avermitilis: deducing the ability of producing secondary metabolites.
Proceedings of the National Academy of Sciences of the United States of America 98,
12215-20.
170
Orjala, J., Nagle, D. G., Hsu, V., and Gerwick, W. H. (1995). Antillatoxin, an
exceptionally ichthyotoxic cyclic lipopeptide from the tropical cyanobacterium
Lyngbya majuscula. Journal of American Chemical Society 117, 8281-8282.
Orjala, J. and Gerwick, W. H. (1996). Barbamide, a chlorinated metabolite with
molluscicidal activity from the Caribbean cyanobacterium Lyngbya majuscula. Journal
of Natural Products 59, 427-43 0.
On, P. T. and Jones, G. J. (1998). Relationship between microcystin production and
cell division rates in nitrogen limited Microcystis aeruginosa cultures. Limnology and
Oceanography 43, 1604-1614.
Osborne, N. J., Webb, P. M., and Shaw, G, R. (2001). The toxins of Lyngbya
majuscula and their human and ecological health effects. Environment International
27, 38 1-392.
Paerl,H.W. (1988). Growth and reproduction strategies of freshwater blue-green algae
(cyanobacteria). In Growth and reproductive strategies of freshwater phytoplankton,
C.D.Sandgren, ed. (Cambridge: Cambridge University Press), pp. 261-3 15.
Paitan, Y., On, E., Ron, E. Z., and Rosenberg, E. (1999a). Cloning and
characterization of a Myxococcus xanthus cytochrome P-450 hydroxylase required for
biosynthesis of the polyketide antibiotic TA. Gene 228, 147-53.
Paitan, Y., On, E., Ron, E. Z., and Rosenberg, E. (1999b). Genetic and functional
analysis of genes required for the post- modification of the polyketide antibiotic TA of
Myxococcus xanthus. Microbiology 145, 3059-3067.
Patterson, G. M. L., Baker, K. B., Baldwin, C. L., Bolis, C. M., Caplan, F. R., Larsen,
L. K., Levine, I. A., Moore, R. E., Nelson, C. S., Tschappat, K. D., Tuang, G. D.,
Boyd, M. R., Cardellina, J. H., Collins, R. P., Gustafson, K. R., Snader, K. M.,
Weislow, 0. S., and Lewin, R. A. (1993). Antiviral activity of cultured blue-green
algae (cyanophyta). Journal of Phycology 29, 125-130.
Patterson, G. M. L., Baldwin, C. L., Bolis, C. M., Caplan, F. R., Karuso, H., Larsen, L.
K., Levine, I. A., Moore, R. E., Nelson, C. S., Tschappat, K. D., Tuang, G. D.,
Furasawa, B., Furasawa, S., Norton, T. R., and Raybourne, R. B. (1991).
Antineoplastic activity of cultured blue-green algae (cyanophyta). Journal of
Phycology 27, 53 0-536.
Patterson, G. M. L. and Bolis, C. M. (1995). Regulation of scytophycin accumulation
in cultures of Scytonema ocellatum. II. Nutrient requirements. Applied Microbiology
and Biotechnology 43, 692-700.
Patterson, G. M. L., Larsen, L. K., and Moore, R. B. (1994). Bioactive natural
products from blue-green algae. Journal of Applied Phycology 6, 15 1-157.
171
Perez-Baz, J., Canedo, L. M., Fernandez Puentes, J. L., and Silva Elipe, M. V. (1997).
Thiocoraline, a novel depsipeptide with antitumor activity produced by a marine
Micromonospora. II. Physico-chemical properties and structure determination. Journal
of Antibiotics (Tokyo) 50, 738-741.
Pettit, G. R., Herald, C. L., Doubek, D. L., Delbert, A. L., Arnold, B., and Clardy, J.
(1982). Isolation and structure of bryostatin 1. Journal of American Chemical Society
104, 6846-6848.
Pettit, G. R., Kamano, Y., Herald, C. L., Tuinman, A. A., Boettner, F. E., Kizu, H.,
Tomer, K. B., and Bontems, R. J. (1987). The Isolation and Structure of a Remarkable
Marine Animal Antineoplastic Constituent: Dolastatin 10. Journal of American
Chemical Society 109, 6883-6885.
Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E., and Khosla, C. (2001).
Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli.
Science 291, 1790-1792.
Pfeifer, B. A. and Khosla, C. (2001). Biosynthesis of polyketides in heterologous
hosts. Microbiology and Molecular Biology Reviews 65, 106-118.
Pfeifer, B. A., Wang, C. C., Walsh, C. T., and Khosla, C. (2003). Biosynthesis of
Yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as
a heterologous host. Applied and Environmental Microbiology 69, 6698-6702.
Piel, J. (2004). Metabolites from symbiotic bacteria. Nat Prod Rep 21, 5 19-538.
Piel, J., Hertweck, C., Shipley, P. R., Hunt, D. M., Newman, M. S., and Moore, B. S.
(2000). Cloning, sequencing and analysis of the enterocin biosynthesis gene cluster
from the marine isolate 'Streptomyces maritimus': evidence for the derailment of an
aromatic polyketide synthase. Chemistry & Biology 7, 943-955.
Pradella, S., Hans, A., Sproer, C., Reichenbach, H., Gerth, K., and Beyer, S. (2002).
Charactensation, genome size and genetic manipulation of the myxobacterium
Sorangium cellulosum So ce56. Archives of Microbiology 178, 484-92.
Proteau, P. 3., Gerwick, W. H., Garcia-Pichel, F., and Castenholz, R. (1993). The
structure of scytonemin, an ultraviolet sunscreen pigment from the sheaths of
cyanobacteria. Experientia 49, 825-829.
Quadri, L. E. N., Keating, T. A., Patel, H. M., and Walsh, C. T. (1999). Assembly of
the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin: In vitro
reconstitution of aryl-4,2-bisthiazoline synthetase activity from PchD, PPchE, and
PchF. Biochemistry 38, 1494 1-14954.
172
Rapala, J., Sivonen, K., Lyra, C., and Niemela, S. I. (1997). Variation of microcystins,
cyanobacterial hepatotoxins, in Anabaena spp. as a function of growth stimuli.
Applied and Environmental Microbiology 63, 2206-2212.
Recktenwald, J., Shawky, R., Puk, 0., Pfennig, F., Keller, U., Wohileben, W., and
Peizer, S. (2002). Nonribosomal biosynthesis of vancomycin-type antibiotics: a
heptapeptide backbone and eight peptide synthetase modules. Microbiology 148,
1105-18.
Reid, R., Piagentini, M., Rodriguez, B., Ashley, G., Viswanathan, N., Carney, J.,
Santi, D. V., Hutchinson, C. R., and McDaniel, R. (2003). A model of structure and
catalysis for ketoreductase domains in modular polyketide synthases. Biochemistry 42,
72-79.
Repka, S., Koivula, M., Harjunpa, V., Rouhiainen, L., and Sivonen, K. (2004). Effects
of phosphate and light on growth of and bioactive peptide production by the
Cyanobacterium anabaena strain 90 and its anabaenopeptilide mutant. Applied and
Environmental Microbiology 70, 4551-4560.
Rinehart, K. L., Holt, T. G., Fregeau, N., Stroh, J. G., Keifer, P. A., Sun, F., Li, H. L.,
and Martin, D. G. (1990). Ecteinascidins 729, 743, 745, 759A, 759B, and 770: potent
antitumor agents from the Caribbean tunicate Ecteinascidia turbinata. Journal of
Organic Chemistry 55, 4512-4515.
Rinehart, K. L. and Lithgow-Bertelloni, A. M. Dehydrodidemnin B. WO 1990GB1495[WO 9104985], 1-41. 1991.
Ref Type: Patent
Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., and Stainier, R. Y. (1971).
Generic assignments, strain histories and properties of pure cultures of cyanobacteria.
Journal of General Microbiology 111, 1-61.
Romero, F., Espliego, F., Perez-Baz, J., de Quesada, G., Gravalos, D., De Ia Calle, F.,
and Fernandez Puentes, J. L. (1997). Thiocoraline, a new depsipeptide with antitumor
activity produced by a marine Micromonospora. I. Taxonomy, fermentation, isolation,
and biological activities. Journal of Antibiotics (Tokyo) 50, 734-737.
Rose, T. M., Schultz, E. R., Henikoff, J. G., Pietrokovski, S., McCallum, C. M., and
Henikoff, S. (1998). Consensus-degenerate hybrid oligonucleotide primers for
amplification of distantly related sequences. Nucleic Acids Research 26, 1628-35.
Rossi, J. V., Roberts, M. A., Yoo, H.-D., and Gerwick, W. H. (1997). Pilot scale
culture of the marine cyanobacterium Lyngbya majuscula for its pharmaceuticallyuseful natural metabolite curacin A. Journal of Applied Phycology 9, 195-204.
173
Rouhiainen, L., Paulin, L., Suomalainen, S., Hyytiainen, H., Buikema, W., Haselkorn,
R., and Sivonen, K. (2000). Genes encoding synthetases of cyclic depsipeptides,
anabaenopeptilides, in Anabaena strain 90. Molecular Microbiology 37, 156-67.
Salomon, C. E., Magarvey, N. A., and Sherman, D. H. (2004). Merging the potential
of microbial genetics with biological and chemical diversity: an even brighter future
for marine natural product drug discovery. Natural Product Reports 21, 105-12 1.
Sambrook,J., Fritsch,E.F., and Maniatis,T. (1989). Molecular Cloning: A Laboratory
Manual. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).
Sandmann, A., Sasse, F., and Muller, R. (2004). Identification and analysis of the core
biosynthetic machinery of tubulysin, a potent cytotoxin with potential anticancer
activity. Chemistry & Biology 11, 1071-1079.
Schopf,J.W. (2005). The fossil record: Tracing the roots of cyanobacterial lineage. In
The ecology of cyanobacteria: Their diversity in time and space, B.A.Whitton and
M.Potts, eds. (Netherlands: Kiuwer Academic Publishers), pp. 13-35.
Schneider, T. L., Shen, B., and Walsh, C. T. (2003). Oxidase domains in epothiolone
and bleomycin biosynthesis: thiazoline to thiazole oxidation during chain elongation.
Biochemistry 42, 9722-973 0.
Schwartz, R. E., Hirsch, C. F., Sesin, D. F., Flor, J. E., Chartrain, M., Fromtling, R. E.,
Harris, G. H., Salvatore, M. J., Liesch, J. M., and Yudin, K. (1990). Pharmaceuticals
from cultured algae. Journal of Industrial Microbiology 5, 113-124.
Schwarzer, D., Finking, R., and Marahiel, M. A. (2003). Nonribosomal peptides: from
genes to products. Natural Product Reports 20, 275-287.
Schwecke, T., Aparicio, J. F., Molnar, I., Konig, A., Khaw, L. E., Haydock, S. F.,
Oliynyk, M., Caffrey, P., Cortes, J., Lester, J. B., and et at. (1995). The biosynthetic
gene cluster for the polyketide immunosuppressant rapamycin. Proceedings of the
National Academy of Sciences of the United States of America 92, 7839-7843.
Seo, P.-L. and Yokota, A. (2003). The phylogenetic relationships of cyanobacteria
inferred from 1 6S rRNA, gyrB, rpoCl and rpoDl gene sequences. Journal of General
and Applied Microbiology 49, 191-203.
Sharkey, F. H., Banat, I., and Marchant, R. (2004). Detection and quantification of
gene expression in environmental bacteriology. Applied and Environmental
Microbiology 70, 3795-3806.
Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T.,
Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H. (2002). Complete genome
174
sequence of Clostridium perfringens, an anaerobic flesh-eater. Proceedings of the
National Academy of Sciences of the United States of America 99, 996-1001.
Sieber, S., Linne, U., Hilison, N., Roche, B., Walsh, C., and Marahiel, M. (2002).
Evidence for a monomeric structure of nonribosomal Peptide synthetases. Chemistry
& Biology 9, 997.
Sieber, S. A. and Marahiel, M. A. (2005). Molecular mechanisms underlying
nonribosomal peptide synthesis: approaches to new antibiotics. Chemical Reviews
105, 715-738.
Sieber, S. A. and Marahiel, M. A. (2003). Learning from Nature's drug factories:
Nom-ibosomal peptide synthesis of macrocyclic peptides. Journal of Bacteriology 185,
7036-7043.
Sieber, S. A., Walsh, C. T., and Marahiel, M. A. (2003). Loading peptidyl-coenzyme
A onto peptidyl carrier proteins: A novel approach in characterizing macrocyclization
by thioesterase domains. Journal of American Chemical Society 125, 10862-10866.
Silakowski, B., Kunze, B., Nordsiek, G., Blocker, H., Hofle, G., and Muller, R.
(2000). The myxochelin iron transport regulon of the myxobacterium Stigmatella
aurantiaca Sg a15. European Journal of Biochemistry 267, 6476-6485.
Silakowski, B., Schairer, H. U., Bhret, H., Kunze, B., Weinig, S., Nordsiek, G.,
Brandt, P., Blocker, H., Hofle, G., Beyer, S., and Muller, R. (1999). New lessons for
combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene
cluster of Stigmatella aurantiaca DW4/3-1. Journal of Biological Chemistry 274,
37391-37399.
Silakowski, B., Kunze, B., and Muller, R. (2001). Multiple hybrid polyketide
synthase/non-ribosomal peptide synthetase gene clusters in the myxobacterium
Stigmatella aurantiaca. Gene 275, 233-240.
Silby, M. W. and Mahanty, H. K. (2000). The importance of recA mutant strains for
the study of antifungal genes in Pseudomonas aureofaciens PA147-2. Canadian
Journal of Microbiology 46, 744-74 8.
Simmons, T. L., Adrianasolo, E., McPbail, K., Flatt, P., and Gerwick, W. H. (2005).
Marine natural products as anticancer drugs. Mol Cancer Ther 4, 333-342.
Sings, H. L. and Rinehart, K. L. (1996). Compounds produced from potential tunicateblue-green algal symbiosis: a review. Journal of Industrial Microbiology 17, 385-396.
Sitachitta, N., Rossi, J., Roberts, M. A., Gerwick, W. H., Fletcher, M. D., and Willis,
C. L. (1998). Biosynthesis of the marine cyanobacterial metabolite barbamide. 1.
175
Origin of the trichioromethyl group. Journal of American Chemical Society 120,
7131-7132.
Solomon, A. B. and Stoughton, R. B. (1978). Dermatitis from purified sea algae toxin
(debromoaplysiatoxin). Archives of Dermatology 114, 1333-5.
Sone, H., Kondo, T., Kiruyu, M., Ishiwata, H., Ojika, M., and Yamada, K. (1995).
Dolabellin, a Cytotoxic Bisthiazole Metabolite from the Sea Hare Dolabella
auricularia: Structural Determination and Synthesis. Journal of Organic Chemistry 60,
4774-4781.
Song, J., Dong, F., Lilly, J. W., Stupar, R. M., and Jiang, J. (2001). Instability of
bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA
sequences. Genome 44, 463-469.
Stachelhaus, T., Mootz, H. D., and Marahiel, M. A. (1999). The specificity-conferring
code of adenylation domains in nonribosomal peptide synthetases. Chemistry &
Biology 6, 493-505.
Stachelhaus, T. and Marahiel, M. A. (1995). Modular structure of peptide synthetases
revealed by dissection of the multifunctional enzyme GrsA. Journal of Biological
Chemistry 270, 6163-6169.
Stanier, R. Y., Sistrom, W. R., Hansen, T. A., Whitton, B. A., Castenholz, R. W.,
Pfenning, N., Gorlenko, V. N., Kondratieva, E. N., Eimhjellen, K. B., Whittenbury, R.,
Gherna, R. L., and Trtiper, H. G. (1978). Proposal to place the nomenclature of the
cyanobacteria (blue-green algae) under the rules of the International Code of
Nomenclature of Bacteria. International Journal of Systematic Bacteriology 28, 335336.
Staunton, J., Caffrey, P., Aparicio, J. F., Roberts, G. A., Bethell, S. S., and Leadlay, P.
F. (1996). Evidence for a double-helical structure for modular polyketide synthases.
Nature Structural Biology 3, 188-92.
Staunton, J. and Weissman, K. J. (2001). Polyketide biosynthesis: a millennium
review. Natural Product Reports 18, 380-416.
Steege, D. A. (2000). Emerging features of mRNA decay in bacteria. RNA 6, 10791090.
Steffensky, M., Muhienweg, A., Wang, Z. X., Li, S. M., and Heide, L. (2000).
Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides
NCIB 11891. Antimicrobial Agents and Chemotherapy 44, 1214-1222.
Stevenson, C. S., Capper, E. A., Roshak, A. K., Marquez, B., Eichrnan, C., Jackson, J.
R., Mattern, M., Gerwick, W. H., Jacobs, R. S., and Marshall, L. A. (2002a). The
176
identification and characterization of the marine natural product scytonemin as a novel
antiproliferative pharmacophore. Journal of Pharmacology and Experimental
Therapeutics 303, 858-866.
Stevenson, C. S., Capper, E. A., Roshak, A. K., Marquez, B., Grace, K., Gerwick, W.
H., Jacobs, R. S., and Marshall, L. A. (2002b). Scytonemin--a marine natural product
inhibitor of kinases key in hyperproliferative inflammatory diseases. Inflammation
Research 51, 112-114.
Symmank, H., Saenger, W., and Bernhard, F. (1999). Analysis of engineered
multifunctional peptide synthetases - Enzymatic characterization of surfactin
synthetase domains in hybrid bimodular systems. Journal of Biological Chemistry
274, 21581-21588.
Takashima, M. and Sakai, H. (1960). A new toxic substance, teleocidin, produced by
Streptomyces. II. Biological studies of teleocidin. Bulletin of the Agricultural
Chemical Society of Japan 24, 652-655.
Tang, L., Shah, S., Chung, L., Camey, J., Katz, L., Khosla, C., and Julien, B. (2000).
Cloning and heterologous expression of the epothilone gene cluster. Science 287, 640642.
Taraboletti, G., Poli, M., Dossi, R., Manenti, L., Borsotti, P., Faircloth, G., Broggini,
M., D'Inalci, M., Ribatti, D., and Goavazzi, R. (2004). Antiangiogenic activity of
aplidine, a new agent of marine origin. Bristish Journal of Cancer 90, 2418-2424.
Thacker, R. W. and Paul, V. J. (2004). Morphological, chemical and genetic diversity
of the tropical cyanobacteria Lyngbya spp. and Symploca spp. (Oscillatoriales).
Applied and Environmental Microbiology 70, 3305-33 12.
Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids
Research 22, 4673-4680.
Tillett, D., Dittmami, E., Erhard, M., von Dohren, H., Bomer, T., and Neilan, B. A.
(2000). Structural organization of microcystin biosynthesis in Microcystis aeruginosa
PCC7806: an integrated peptide-polyketide synthetase system. Chemistry & Biology
7, 753-764.
Tillett, D. and Neilan, B. A. (2000). Xanthogenate nucleic acid isolation from cultured
and environmental cyanobacteria. Journal of Phycology 36, 25 1-258.
Towle, M. J., Salvato, K. A., Budrow, J., Wels, B. F., Kuznetsov, G., Aalfs, K. K.,
Welsh, S., Zheng, W., Seletsk, B. M., Palme, M. H., Habgood, G. J., Singer, L. A.,
DiPietro, L. V., Wang, Y., Chen, J. J., Quincy, D. A., Davis, A., Yoshimatsu, K.,
177
Kishi, Y., Yu, M. J., and Littlefield, B. A. (2001). In vitro and in vivo anticancer
activities of synthetic macrocyclic ketone analogues of halichondrin B. Cancer
Research 61, 1013-1021.
Trauger, J. W., Kohli, R. M., and Walsh, C. T. (2001). Cyclization of backbonesubstituted peptides catalyzed by the thioesterase domain from the tyrocidine
nonribosomal peptide synthetase. Biochemistry 40, 7092-8.
Trauger, J. W. and Walsh, C. T. (2000). Heterologous expression in Escherichia coli
of the first module of the nonribosomal peptide synthetase for chioroeremomycin, a
vancomycin-type glycopeptide antibiotic. Proceedings of the National Academy of
Sciences of the United States of America 97, 3112-3117.
Turgay, K. and Marahiel, M. A. (1994). A General Approach for Identifying and
Cloning Peptide Synthetase Genes. Peptide Research 7, 238-241.
Ulirich, M. and Bender, C. L. (1994). The biosynthetic gene cluster for coronamic
acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acidactivating enzymes and thioesterases. Journal of Bacteriology 176, 7574-7586.
Varon, M., Fuchs, N., Monosov, M., Tolchinsky, S., and Rosenberg, E. (1992).
Mutation and mapping of genes involved in production of the antibiotic TA in
Myxococcus xanthus. Antimicrobial Agents and Chemotherapy 36, 23 16-2321.
Venter, J. C., Remington, K., Heidelberg, J. F., Halpern, A. L., Rusch, D., Eisen, J. A.,
Wu, D., Paulsen, I., Nelson, K. E., Nelson, W., Fouts, D. E., Levy, S., Knap, A. H.,
Lomas, M. W., Nealson, K., White, 0., Peterson, J., Hoffman, J., Parsons, R., BadenTilison, H., Pfannkoch, C., Rogers, Y. H., and Smith, H. 0. (2004). Environmental
genome shotgun sequencing of the Sargasso Sea. Science 304, 66-74.
Walsh, C. T. (2004). Polyketide and nonribosomal peptide antibiotics: modularity and
versatility. Science 303, 1805-18 10.
Wang, Z. X., Li, S. M., and Heide, L. (2000). Identification of the coumermycin A(1)
biosynthetic gene cluster of Streptomyces rishiriensis DSM 40489. Antimicrobial
Agents and Chemotherapy 44, 3040-3048.
Watanabe, K., Wang, C. C., Boddy, C. N., Cane, D. B., and Khosla, C. (2003).
Understanding substrate specificity of polyketide synthase modules by generating
hybrid multimodular synthases. Journal of Biological Chemistry 278, 42020-42026.
Watanabe, M. F. and Oishi, S. (1985). Effects of environmental factors on toxicity of a
cyanobacterium (Microcystis aeruginosa) under culture conditions. Appi Environ
Microbiol 49, 1342-1344.
178
Weber, G., Schorgendorfer, K., Schneiderscherzer, E., and Leitner, E. (1994). The
Peptide Synthetase Catalyzing Cyclosporine Production in Tolypocladium Niveum Is
Encoded by a Giant 45.8-Kilobase Open Reading Frame. Current Genetics 26, 120125.
Weissman, K. J. (2004). Polyketide biosynthesis: understanding and exploiting
modularity. Philosophical Transactions of the Royal Society of London Series AMath, Physics and Engineering Sciences 362, 267 1-2690.
Whitton,B.A. and Potts,M. (2000). Introduction to cyanobacteria. In The ecology of
cyanobacteria. Their diversity in time and space., B.A.Whitton and M.Potts, eds.
(Dordrecht: Kiuwer Academic Publisher), pp. 1-11.
Williamson, R. T., Boulanger, A., Vulpanovici, A., Roberts, M. A., and Gerwick, W.
H. (2002). Structure and absolute stereochemistry of phormidolide, a new toxic
metabolite from marine cyanobacterium Phormidium sp. Journal of Organic
Chemistry 67, 7927-7936.
Wilmotte,A. (1994). Molecular evolution and taxanomy of cyanobacteria. In The
Molecular Biology of Cyanobacteria, D.A.Bryant, ed. (Netherlands: Kiuwer Academic
Publishers), pp. 1-25.
Wipf, P., Reeves, J. T., and Day, B. W. (2004). Chemistry and biology of curacin A
2. Current Pharmaceutical Design 10, 1417-1437.
Wong, F. C. and Meeks, J. C. (2001). The hetF gene product is essential to heterocyst
differentiation and affects HetR function in the cyanobacterium Nostoc punctifonne.
Journal of Bacteriology 183, 2654-2661.
Wu, M. Novel bioactive secondary metabolites from the marine cyanobacterium
Lyngbya majuscula. 1996. Oregon State University.
Ref Type: Thesis/Dissertation
Wu, M., Okino, T., Nogle, L. M., Marquez, B. L., Williamson, R. T., Sitachitta, N.,
Berman, F. W., Murray, T. F., McGough, K., Jacobs, R., Colsen, K., Asano, T.,
Yokokawa, F., Shioiri, T., and Gerwick, W. H. (2000). Structure, Synthesis, and
Biological Properties of Kalkitoxin, a Novel Neurotoxin from the Marine
Cyanobacterium Lyngbya majuscula. Journal of American Chemical Society 122,
12041-12042.
Wu, N., Tsuji, S. Y., Cane, D. E., and Khosla, C. (2001). Assessing the Balance
between Protein-Protein Interactions and Enzyme- Substrate Interactions in the
Channeling of Intermediates between Polyketide Synthase Modules. Journal of
American Chemical Society 123, 6465-6474.
179
Zazopoulos, E., Huang, K., Staffa, A., Liu, W., Bachmann, B. 0., Nonaka, K., Ahiert,
J., Thorson, J. S., Shen, B., and Farnet, C. M. (2003). A genomics-guided approach for
discovering and expressing cryptic metabolic pathways. Nature Biotechnology 21,
187-190.
Zhang, J. H., Quigley, N. B., and Gross, D. C. (1995). Analysis of the syrB and syrC
genes of Pseudomonas syringae pv. syringae indicates that syringomycin is
synthesized by a thiotemplate mechanism. Journal of Bacteriology 177, 4009-4020.