EU MARIE CURIE: LYNGBYA KENYA - PROJECT PIIF-GA-2011-299550 CYP450 BIOSYNTHESIS OF LYNGBYA MAJUSCULA NATURAL PRODUCTS
FINAL REPORT
SCIENTIFIC COORDINATOR
Professor J GRANT BURGESS
School of Marine Science and Technology
Newcastle University
Armstrong Building, Queen Victoria Road
NE1 7RU
United Kingdom
Tel. +44 (0) 191 2226717
Fax. +44 (0) 191 2225491
Email. grant.burgess@newcastle.ac.uk
THE FELLOW
Dr THOMAS M DZEHA
Department of Chemistry and Biochemistry
Pwani University
P.O. Box 195-80108
Kilifi
Kenya
Tel. +254 (0) 788986063
Email. thomas.dzeha@gmail.com
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EXECUTIVE SUMMARY
A sustainable supply of marine natural products for potential therapeutics is one of the
greatest challenges facing drug discovery efforts today, especially during clinical trials.
Nearly 300 compounds with therapeutic potential have been isolated from the tropical
marine cyanobacterium Lyngbya majuscula. However there are considerable concerns
regarding the real source of this large number of natural products attributed to L. majuscula.
This project focused on the cytochrome p450 biosynthesis of L. majuscula natural products
namely the modular cyclodepsipeptides homodolastatin 16 (HMDS 16) and antanapeptin A
(ANTAP A). L. majuscula was collected from Shimoni, Kenya in April 2012. The aim was to
identify if these compounds originate from the cyanobacteria or bacteria cohabiting with it or
both. Bacteria representing γ-proteobacteria, Firmicutes, and α–proteobacteria were isolated
from the cyanobacteria. Non-ribosomal peptide synthetase (NRPS) and polyketide synthase
(PKS) screens of bacteria with known complete genome sequences using bioinformatics
tools showed in this study that the modular assembly lines for these bacteria are inconsistent
with those of HMDS 16 and ANTAP A. However, Bacillus licheniformis and Marinobacterium
stanieri synthesise the β amino acid dolamethleuline and Klebsiella oxytoca is involved with
the biosynthesis of the unusual dolaphenvaline amino acid in HMDS 16 putatively. Profiling
for the cyclodepsipeptides in bacteria supernatants using liquid chromatograph mass
spectrometry (LCMS) confirmed the presence of HMDS 16, its analogue dolastatin 16, and
ANTAP A in L. majuscula but not in the bacteria. Subsequently such leads aided the
prospects for obtaining the complete genome sequence of L. majuscula from its
metagenome and in identifying gene clusters encoding for HMDS 16 and ANTAP A. The
cloning and heterologous expression of the gene clusters for these especially important
anticancer agents is the goal that we aimed to attain by the end of the program.
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TABLE OF CONTENTS
Page
Summary
2
Table of contents
3
Key words and abbreviations
5
A
SCIENTIFIC REPORT
6
A1
Project context and objectives
6
A2
Modular assembly of homodolastatin 16
9
A3
Investigations of growth conditions for the Kenyan Lyngbya majuscula
10
A 3.1 Growth of L. majuscula in autoclaved seawater
11
A 3.2 Growth of Lyngbya in autoclaved seawater and antibiotics
11
A 3.3 Treatment of Lyngbya majuscula with antibiotics prior to culturing 12
A4
A 3.4 Growth of Lyngbya in autoclaved seawater, BG11 and
KNO3 (15 gL-1)
12
A 3.5 Growth of Lyngbya in autoclaved seawater, BG11, KNO3
and antibiotics
13
A 3.6 Summary of the results of bacteria isolates on Lyngbya
majuscula
13
Isolation of Lyngbya majuscula filaments
14
A 4.1 The ecology of the Kenyan L. majuscula
14
A 4.2 Do dead L. majuscula filaments contain homodolastatin 16?
16
A5
Bioinformatics strategies for identifying homodolastatin 16 gene clusters 17
A6
Significant outcomes of the scientific project
18
A 6.1 Isolation of culturable bacteria co-habiting with L. majuscula
18
A 6.2 Phylogeny of bacterial isolates and related taxon
18
A 6.3 16S rDNA isolation and identification of L. majuscula
and A. colombiense
19
A 6.4 Antibiotic resistant bacteria associated with L. majuscula filament 20
A 6.5 LCMS profiling of homodolastatin 16 in L. majuscula
and epibiotic bacteria
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21
A7
B
A 6.6 Bioinformatics prediction for NRPS modular compounds
22
A 6.7 Putative biosynthesis of dolamethyleuline and
dolaphenvaline fragments
23
A 6.8 Molecular identification of the Kenyan “Lyngbya majuscula”
25
A 6.9 Analysis of sequence data and phylogeny of the Kenya
L. majuscula
27
Manuscripts
28
IMPACT
29
B1
Seminars and guest lectures
29
B2
International conferences and symposia
30
B 2.1 Federation of European Biochemical Society (FEBS) 2013
conference at St. Petersberg, Russia from 6-12 July 2013.
30
B 2.2 International Advanced Single Cell Biotechnology at
Sheffield University, UK on 12 February 2014
32
Outstanding outcomes of the project
32
B 3.1 Grant Applications
32
B 3.2 Outreach program - Mentoring of Marine Biology students
33
B3
C
THE UNITED KINGDOM AND MY EU MARIE CURIE FELLOWSHIP
34
D
ACKNOWLEDGEMENT
34
E
REFERENCES
35
F
ANNEX
37
Annex 1
Table 1 of Kenyan Lyngbya majuscula epibiotic bacteria (EB)
isolates
37
Annex 2:
Phylogeny of EB isolates
38
Annex 3:
Adenylation domain for Moorea producens
39
Annex 4:
MtaD-M1-Cys Biosynthesis
43
Annex 5:
TycA-M1-D-/L-Phe Biosynthesis
44
Annex 6:
List of PARSE HMM modular domains for M. producens
45
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KEY WORDS:
Lyngbya majuscula, Homodolastatin 16, Dolastatin 16, Antanapeptin A, Epibiotic bacteria,
16S rDNA, NRPS/PKS, gene cluster, modular assembly, LCMS, putative biosynthesis,
phylogeny, copper sulfate, molecular identification and differential DNA isolation
ABBREVIATIONS
A - adenylation
ANTAP A – Antanapeptin A
AT - acyl transferase
Cy – cyclisation
DH – Dehydratase
DML – Dolamethleuline
DPV – Dolaphenvaline
ER - enoyl reductase
HIV – Hydroxyisovaleriate
HMDS 16 – Homodolastatin 16
KR – Ketoreductase
KS - ketosynthase
LCMS – Liquid chromatograph mass spectrometry
M - Methylation
NMe-Ile – N-methylisoleucine
NRPS – Non-ribosomal peptide synthetase
Phe – Phenylalanine
PKS – Polyketide synthase
T- thiolation
TE - Thioesteration
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A
SCIENTIFIC REPORT
A1
Project context and objectives
The marine biotope has been identified as a large and rich area for exploration of biologically
active pharmaceuticals of use in medicine and in biotechnology. The expansive diversity in
form and function of the marine environment coupled with the unique adaptations of the
marine organisms therein and varied biosynthesis pathways compared with the terrestrial
world suggest that it is as yet an untapped resource. Nearly 15 marine natural products are
in various phases of clinical development, mainly in oncology, with several products already
on the market and with more on the way.1
Research over the last four decades has shown the filamentous marine cyanobacterium,
Lyngbya majuscula of the order Oscillatoriales to be a prolific source of a diverse range of
modular natural products
2-4
Out of the nearly 800 compounds isolated from marine
cyanobacteria, L. majuscula dominates with nearly 300 substances coming from the
species.5 The plethora of marine derived natural products isolated from L. majuscula
worldwide in pan-tropically geographical locations include those which exhibit antimicrobial
activity, anti-proliferative compounds, anti-HIV agents and those which have shown potential
as anticancer agents.5,6 Useful L. majuscula natural products include the anticancer agent
Curacin A, the neurotoxic jamaicamides and the UV-sunscreen pigment scytonemin.7
Investigations into the biosynthesis of L. majuscula natural products have revealed gene
clusters encoding modular, mixed polyketide synthase (PKS)/nonribosomal peptide
synthetase assembly lines that incorporate other functional groups through highly unusual
mechanisms.3,4 Homodolastatin 16 (1) and antanapeptin A (2) isolated from the Kenyan L.
majuscula exemplify such assemblies.2 The former is a mixed NRPS/PKS modular
cyclodepsipeptide isolated from the Kenyan marine cyanobacterium L. majuscula along with
antanapeptin A (2) and has moderate activity towards the oesophageal cancer cell lines
WHCO1 and WHCO6 (IC50 values of 4.3 and 10.1 μg/mL respectively).2 Its analogue
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dolastatin 16 (3) shows very strong activity against lung (NCI-H460: GI50 0.00096 μg mL-1),
colon (KM20L2 GI50 0.0012 μgmL-1), brain (SF-295, GI50 0.0052 μgmL-1) and melanoma (SKMEL5 GI50 0.0033 μgmL-1) cancer cell lines.8,9
1 R = N-Me-Ile
2
3 R = N-Me-Val
The biological activities exhibited by homodolastatin 16 (1) and its analogue dolastatin 16 (3)
towards cancer cell lines suggests that there is a need to obtain sustainable amounts of
these modular compounds for further investigations including structure activity relationships
and clinical testing. A sustainable supply of these cyclodepsipeptides could only be achieved
through aquaculture of the organism, chemical synthesis or by recombinant biosynthesis of a
source organism.10 A complex circadian rhythm associated with cyanobacteria rules out
aquaculture as a possible alternative.11 Low enantiomeric excess (e.e) yields and refractory
problems associated with chemical synthesis only suggest that recombinant biosynthesis of
these natural products is the ideal way to realizing sustainability. Gene shuffling, domain
deletions and mutations are recombinant biosynthesis engineering tools that are currently
used for the identification and cloning of gene clusters for polyketides, NRPS and hybrid
polyketide-NRP metabolites.12
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Recently in 2011, the genome of Lyngbya majuscula 3L which is a Caribbean strain that
produces the tubulin polymerization inhibitor curacin A and the molluscicide barbamide was
sequenced using Sanger and 454 sequencing approaches to near completion.3 Whereas the
draft genome sequence revealed gene clusters for Curacin A and barbamide, only a mere
3% of the genes were dedicated to secondary metabolite production, biosynthesis, transport
and catabolism.3 Questions therefore arise as to whether or not the natural products are
strain specific, especially as most taxonomic classifications have been mostly morphological.
It is also clear through the draft genome sequence of Lyngbya majuscula 3L that the
cyanobacterium not only lacks the necessary nifH gene for photosynthesis but also encodes
for a complex gene regulatory system for microbial association and environmental
adaptation. Distinctively, there is a paucity of information regarding if the compounds
isolated from L. majuscula originate from the cyanobacterium or the bacteria co-habiting with
it or both, especially as efforts to render axenic L. majuscula culturable have been mostly
fruitless. Subsequently there are considerable concerns regarding the real source of the
large number of natural products attributed to L. majuscula.
Given the biological activities of homodolastatin 16 (1) and its analogue dolastatin 16 (3)
towards cancer cell lines, investigations to identify which of bacteria or the non-axenic
filamentous
cyanobacterium
produces
the
cyclodepsipeptides
were
necessary.
Subsequently, we report the 16S rDNA isolation and identification of bacteria cohabiting with
the Kenyan L. majuscula; bacteria found on the filament and the LCMS profiling for
homodolastatin 16 (1), antanapeptin A (2) and dolastatin 16 (3) in organic extracts of L.
majuscula and bacterial isolates supernatants. For clarity putative probing of gene clusters of
these modular compounds in the genomes of bacteria closely associated with the Kenyan L.
majuscula in databases was carried out. Further, we report a new method for identifying
non-axenic cyanobacteria. These findings have important implications on the understanding
of symbiotic pathways for L. majuscula and in the recombinant biosynthesis of
homodolastatin 16 (1) and its potent anticancer analogue dolastatin 16 (3).
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SCIENTIFIC AND TECHNOLOGICAL RESULTS
A2
Modular Assembly of homodolastatin 16
A thorough understanding of the modular assembly of homodolastatin 16 was essential in
gaining insight into the biosynthesis pathway mechanism of the cyclodepsipeptide. The
assembly could be inferred by examining the structure of the cyclodepsipepide. The
structure comprises 3 proline moieties, an n-methyl leucine, a hydroxyisovaleriate (HIV) and
two unusual beta-hydroxy amino acids, namely dolaphenvaline (dpv) and dolamethleuline
(dml). We proposed the following putative modular assembly for homodolastatin 16:
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Homodolastatin 16 above is likely to use the following building blocks; the beta methyl homo
Phe (DPV) probably coming from methylation of the alpha keto acid, then transamination to
beta methyl Phe then chain followed by extension as is Ileu biosynthesis to yield the
homoskeleton. The Lactate and HIV hydroxy acids may be activated by keto acidrecognizing A domains with an embedded downstream NADH-dependent dehydrogenase in
the module. The DML residue almost certainly uses a hybride NRPS-PKS module
condensing Val and Methyl malonyl CoA. This PKS module should have KR/DH/ER domain
to take the initial tethered beta keto extended Val-Me-mal scaffold to the fully saturated one
here. This is precedent in statine assembly. The gamma amino group that results is the key
indicator.
In our quest to further understand the modular assembly and to identify the gene clusters for
homodolastatin 16, we looked at compounds with similar unusual beta-amino acid fragments
and came up with the following literature: the isolation from cephalaspidean mollusk
Philinopsis speciosa and structure elucidation of kulokekahilide;13 and the isolation from
cyanobacterium Lyngbya majuscula and structure elucidation of pitiprolamide.14
A3
Investigations of growth conditions for the Kenyan Lyngbya majuscula
Lyngbya majuscula was investigated for growth conditions to establish how to render it
cultivable. It should be noted that there has been controversy regarding whether or not the
cyanobacteria possesses the niFH gene for photosynthesis. Lundgren et al. in 2003 reported
the niFH gene for photosynthesis in L. majuscula on the basis of the acetylene reduction
assay. Studies by Gerwick and co-workers on the near complete genome of Moorea
producens (L. majuscula), assert that the cyanobacterium does not contain niFH genes in its
genome but is endowed with substantial amount of genes for microbial association. In our
study we hypothesized that if any genes for photosynthesis were observed in the acetylene
reduction studies, they may have been attributed to association especially by nitrogen fixing
bacteria associated with L. majuscula. The following experiments were therefore designed to
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address the question as to why it is rather difficult to grow L. majuscula under ordinary
laboratory conditions.
A 3.1 Growth of L. majuscula in autoclaved seawater
Lyngbya majuscula was grown in autoclaved seawater from 5th March 2013 with unchanged
medium until 17th April 2013 (1 month 2 weeks) in an orbital shaker incubator (27 °C, mild
constant light). Observations revealed the cyanobacterium to be unhealthy and lacked the
green pigmentation for photosynthesis. Isolation of bacteria from the Lyngbya mat grown
under these conditions highlighted the following observations:
Two main bacteria species were isolated from the medium namely the bright orange
Shewanella algae sp. and the crusty creamy Klebsiella oxytoca. These were replicated from
an isolation of bacteria from the L. majuscula mat. Re-culturing of the cyanobacteria for a
further two weeks (17th to 30th April) under the same conditions but with fresh autoclaved
seawater media resulted into further degeneration of the cyanobacterium, totally lacking in
growth prospects and exhibiting no signs of the green pigment for photosynthesis. When the
culture was transferred to the refrigerator (4 °C, 2 weeks) regeneration was observed but
when transferred to an open well-lighted environment severe deterioration was observed.
This confirmed the necessity of a light and dark regime for the effective growth of the
cyanobacteria.
A 3.2 Growth of Lyngbya in autoclaved seawater and antibiotics
It was desired to make the cyanobacteria as axenic as possible and also to investigate if the
absence of bacteria had an effect on the growth of L. majuscula. A cocktail of antibiotics
targeting both Gram +Ve and Gram –Ve was made comprising of penicillin, Streptomycin
and chloramphenicol each at the concentration of 4 mg L-1. The antibiotic treated contents
were shaken vigorously to ensure uniform distribution and the cyanobacteria grown (5th
March to 17 April, 2013) without any medium change. It was observed that there was slight
improvement on the growth of L. majuscula compared with the autoclaved seawater alone
11 | P a g e
with some areas showing the green pigmentation for photosynthesis. On this basis we
speculated that bacteria affecting the cyanobacteria negatively were absent. Isolation of
bacteria from the medium and cyanobacteria respectively led to the following observations:
Bright orange (Shewanella algae sp.) and bright yellow (Pseudomonas stutzeri) were
present along with the glassy Pseudomonas putida in the medium, results which were
replicated from an isolation of the Lyngbya mat. However, the abundance of the bacteria on
the plate was a lot less compared with the medium.
A 3.3 Treatment of Lyngbya majuscula with antibiotics prior to culturing
Based on the above observations, it was desired to treat the Lyngbya with the cocktail of
antibiotics out of the medium. The intent was to establish antibiotic resistant bacteria and
whether or not the surviving bacteria are associated with the non-ribosomal peptide
synthetase (NRPS) genes for antibiotics. Treatment of the Lyngbya led to the following
observations:
Consistently, the yellow Pseudomonas stutzeri and the creamy Klebsiella oxytoca and the
greenish Shewanella algae sp. dominated the isolation. Genome mining of the KEGG
genome database revealed that K. oxytoca possessed the non-ribosomal peptide synthetase
(nrps) genes for rifamycin and related antibiotics.
A 3.4 Growth of Lyngbya in autoclaved seawater, BG11 and KNO3 (15 gL-1)
The North Sea water at Newcastle upon Tyne is poor in nitrogen and it was therefore
considered necessary to supplement the autoclaved seawater with nitrogen nitrate from
KNO3 (15g L-1). In common with other cyanobacteria, Lyngbya is generally acknowledged to
grow under low phosphorus phosphate conditions and therefore it was not grown the
Lyngbya under phosphate conditions. Growth from (17th to 30th April) showed improvement
compared with the autoclaved seawater only but comparable to the growth of the antibiotic
treated Lyngbya. Isolation of bacteria from the medium and Lyngbya mat respectively
demonstrated that P. stutzeri, S. algae sp. and K. oxytoca were the key bacteria closely
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associating with L. majuscula. No new recruitment of bacteria was observed by the
incorporation of KNO3 into the medium.
A 3.5 Growth of Lyngbya in autoclaved seawater, BG11, KNO3 and antibiotics
The effect of antibiotics was determined for the growth of Lyngbya in the above medium.
Growth of the cyanobacteria was comparable to that of the Lyngbya in autoclaved seawater,
BG11 and KNO3. Whereas Shewanella algae sp. was absent from the bacterial isolates in
the medium and Lyngbya mat respectively, P. stutzeri and K. oxytoca flourished.
It was noted that regardless of the regeneration due to KNO3, and/or incorporation of the
antibiotic cocktail the cyanobacteria could not survive under constant light.
A3.6
Summary of the results of bacteria isolates on Lyngbya majuscula
Bacteria and condition of Lyngbya
Autoclaved seawater
Shewanella algae sp. (Bright orange)
Lyngbya condition poor
Autoclaved seawater and antibiotics
Shewanella
algae
sp,
Pseudomonas
stutzeri and Klebsiella oxytoca
Lyngbya fair growth
Autoclaved seawater, BG11 and KNO3
P. stutzeri, S. algae sp. and K. oxytoca.
Lyngbya growth good
Autoclaved seawater, BG11, KNO3, and P. stutzeri and K. oxytoca
antibiotics
Lyngbya
medium)
13 | P a g e
Lyngbya growth good
and
antibiotics
only
(no P. stutzeri and K. oxytoca
Lyngbya growth good
A4
Isolation of Lyngbya majuscula filaments
Lyngbya majuscula was treated with cycloheximide overnight (5 mg L-1) to rid it of eukaryotic
cells, protozoa and fungi, rinsed several times with filtered sterile seawater (12 times, 45 μ)
and thereafter left in phosphate poor autoclaved seawater overnight. Following this, the
cyanobacterium was submerged in phosphate buffered saline to detach filaments. The
filaments were thoroughly rinsed, pooled together and weighed to afford 0.25 mg of biomass
for DNA extraction and subsequent genome sequencing. In another experiment, bacteria
were isolated from the filament and plated on marine agar to determine the assemblage of
culturable bacteria associated with the filaments outside the sheath of bacteria. These
experiments were repeated for a near dead Lyngbya mat. Bacteria isolated from the live and
dead bacteria were designated as LFB and DFB respectively. Additional filaments from the
live and near dead Lyngbya were thoroughly washed and stained with acridine and negrosin
to establish association of the filaments with heterotrophic bacteria.
The following observations were made:
Predominantly, a bacteria species with a red tinge at the centre that could not be re-isolated
into a single strain was isolated from the filaments of the live and near dead Lyngbya.
Additionally, a creamy bacteria species characteristic of K. oxytoca was observed. The pcr
amplification of the red bacteria did not generate any 16S rDNA sequence as expected.
A 4.1 The ecology of the Kenyan L. majuscula
In the quest to establish if L. majuscula filaments could entirely be rid of bacteria, filaments
prepared as described previously were stained with acridine to establish cyanobacteriabacteria association of the filament (Fig. 1) and with negrosin to study cell wall and DNA
degradation of the filament by bacteria (Fig. 2).
14 | P a g e
Fig.1. Bacteria on the surface of a live
L. majuscula filament
Fig. 2. DNA (at the centre) and cell wall
material (outside)
Clearly, despite treatment of the cyanobacteria with cycloheximide and several rinses of the
bacteria with phosphate buffered saline (PBS), microscopy (Fig. 1) and the isolation of the
LFB and DFB bacteria from the filament surface revealed that bacteria are always
associated with Lyngbya. This was regardless of the cyanobacteria getting actively involved
with phototropism or on the verge of dying. It is widely acknowledged that oxygen is
poisonous towards cyanobacteria including L. majuscula. Bacteria utilise oxygen for
respiration and cyanobacteria capture carbon dioxide to undergo auto-photosynthesis
creating an energy balance on the cyanobacteria-bacteria interface. Conclusively, it was not
surprising that certain species of bacteria were found inside the cyanobacteria sheaths and
on the filaments for this reason.
Fig. 3. A non-uniform near dead filament
Fig. 4. Bacteria embeded onto surface
of a dying filament
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The green and the brown are the live and dead parts respectively of the filament.
The congregation of live bacteria on the dead filament is shown by the red spots.
We further aimed to monitor the behavior of bacteria on the surface of an untreated filament
that was left overnight to die. Whereas it was observed that the bacteria did not enter the
core of the filament targeting the DNA material, nevertheless there was considerable loss of
cell wall material (Fig.3.) leading to the speculation that some of the bacteria on the surface
survive on organic carbon from the cyanobacteria. These findings were corroborated by a
broken cell wall as observed on a negrosin stained filament similarly left to die overnight.
The bacteria isolated from the sheath and on the filaments of L. majuscula are close
relatives of human pathogenic bacteria suggesting that they may not necessarily be
pathogenic to it. These pathogenic bacteria especially K. oxytoca have been shown through
genome mining to possess the niki B gene for nikimycin and rifamycin respectively. This
suggests that within the consortium some bacteria species in addition to other roles supply
chemical defense arsenals to the host substrate. However it is unclear how the live filament
retains its cylindrical shape despite the presence of a myriad species of bacteria some of
which are cellulose degraders as exhibited by the bacteria on a dying filament (Fig. 4).
A 4.2 Do dead L. majuscula filaments contain homodolastatin 16?
We considered that a comparison of the homodolastatin 16 content of dead and live L.
majuscula filaments would provide a clue on the role of surface bacteria towards the
biosynthesis of homodolastatin 16. The absence of the natural product in a dead filament
would suggest that bacteria have a significant role towards the biosynthesis of
homodolastatin 16. Cycloheximide treated filaments thoroughly cleaned with PBS were left
to die naturally in a sterile container (2 days, ambient temperature) and extracted with
dichloromethane:methanol 2:1 according to the method of Gerwick and co-workers with
16 | P a g e
modifications. The LCMS profiles of the C-18 eluants of the extracts of these dead filaments
compared favorably with those of live filaments.
A5
Bioinformatics strategies for identifying homodolastatin 16 gene clusters
Our overall objective for the project was to identify gene clusters encoding for
homodolastatin 16 for expression in a Saccharomyces cerevisiae vector for a sustainable
supply of the anti-cancer homodolastatin 16 and antanapeptin A cyclodepsipeptides. We
were cognizant of the fact that a near complete genome of L. majuscula, otherwise renamed
Moorea producens 3L because of issues of identity had been accomplished in 2011 by
Gerwick and co-workers at the Scripps Institution of Oceanography, San Diego, La Jolla,
USA. We therefore sought to identify non-ribosomal peptide synthetase (NRPS) domains in
the genome of M. producens as a starting point for our understanding of the biosynthesis of
homodolastatin 16 pathway.
A blast of the near complete genome for M. producens against the NRPase HMM from Pfam
(see http://pfam.sanger.ac.uk/family/PF08415) found 3 hits, two above cut-offs. These were
identified as Pseudomonas pseudoalcaligenes CECT 5344, Klebsiella oxytoca KCTC 1686
Stenotrophomonas maltophilia K279a. Incidentally all three species of bacteria were isolated
from the Kenyan L. majuscula in the current project. A blast of the peptide sequence for M.
producens (see FASTA sequence annex 3) revealed the protein of 10623 amino acids to
comprise a total of 38 domains of which 4 were of adenylation. Two of these domains
encoded for the MtaD-M1-Cyst biosynthesis characteristic of Myxothiazol synthetase,
epothilone synthetase, Bacitracin synthetase and Yersiniabactin synthetase (Annex 4). Each
of these nrps natural products are synthesised from the DLYNLSLI modular assembly. The
third domain had a DAWTVAAV modular assembly predicting for the TycA-M1-D-/L-Phe
biosynthesis (Annex 4). This binding pocket putatively expresses the tyrocidine synthetase,
Gramicidin synthetase and Bacitracin synthetase (Annex 4). The fourth adenylation domain
was thought to comprise a hypothetical protein. An examination of the PARSE HMM hits for
17 | P a g e
M. producens (Annex 5) revealed the adenylation (A), thiolation (T), methylation (M),
thioesteration (TE), enoyl reductase (ER), ketosynthase (KS), acyl transferase (AT),
dehydratase (DH) and cyclisation (Cy) domains.
A6
Significant outcomes of the scientific project
A 6.1 Isolation of culturable bacteria co-habiting with L. majuscula
Direct streaking of bacterial isolates from the Kenyan L. majuscula biomass onto marine
agar 2216 (10% w/v) consistently led to the isolation of colored colonies that were subcultured to obtain pure strains. The concentration of marine agar was altered between 1%
and 10% to differentiate between bacteria growing under poor and rich nutrient medium
respectively. Pseudomonas stutzeri (yellow) was isolated from the sub-culturing of colonies
embedded into Enterobacter cloae (creamy), a mixture that was characterized by a green
pigment. There were diverse morphologies of the bacteria isolates after overnight incubation
including tiny colonies of Bacillus subtilis, glassy Pseudomonas putida and the large soft
Shewanella algae (purple) and Pseudomonas stutzeri (yellow) respectively. Hard colonies
were observed in Bacillus licheniformis (red). A list of the bacteria identified by 16S rDNA is
shown in Table 1. Nearly 70% of all isolates were γ-proteobacteria. Firmicutes were isolated
in reasonable quantities (17%) whereas α-proteobacteria and Actinobacteria were minimal.
A 6.2 Phylogeny of bacterial isolates and related taxon
The evolutionary history of bacteria isolates from L. majuscula investigated along other taxon
of pathogenic bacteria and cyanobacteria using the Maximum Parsimony method shows the
α-proteobacteria
Ochrobactrum
anthropi,
Aminobacterium
colombiense
and
the
actinobacteria Cellulosimicrobium cellulans as being close relatives of L. majuscula (Annex.
2). The mycobacterium A. colombiense was isolated from L. majuscula gDNA as a
consequence of
the cross-reaction with
the cyanobacteria primers.
Surprisingly,
Pseudoalteromonas carrageenovora isolated from the L. majuscula filament appears to have
no close relationship to the cyanobacteria. This finding is corroborated with Klebsiella
18 | P a g e
oxytoca, Shewanella algae species and Pseudomonas stutzeri that associate closely with L.
majuscula. Pseudomonas pseudoalcaligenes related closely with the known pathogen
Pseudomonas tolaasii and Enterobacter cloacae had close relationship with the pathogen
Pseudomonas fluorescens. Klebsiella oxytoca related well with Enterobacter cancerogenous
and Yorkenella regensburgeii. The firmicutes all related fairly well to each other.
A 6.3 16S rDNA isolation and identification of L. majuscula and A. colombiense
The appearance and morphology of the L. majuscula was consistent with the earlier
identification of the homodolastatin 16 producing strain.2 16S rDNA was used to confirm the
identity of the cyanobacterium. In order to achieve quality genomic DNA for the identification,
L. majuscula in filtered (22 μm) autoclaved seawater poor in phosphate phosphorus was
treated with cycloheximide (4 mg mL-1, 12 hrs) to rid it of eukaryotic organisms and
thereafter left submerged in phosphate buffered saline (PBS, pH 7.4, overnight) to detach
the filaments and to remove extracellular polysaccharides.15 The filament remained
associated with bacteria even after several attempts to wash it with PBS and milliQ water
(Fig. 1). Species identification under the microscope was not possible.
Bacteria on the surface of L. majuscula were killed by exposure of the cyanobacterium to
copper sulfate pentahydrate (5 min, 10 min, 30 min and 60 min) prior to weighing aliquots
(0.5g) for DNA extraction. Most bacteria were dead within minutes whilst a few embedded
themselves into the L. majuscula filament tissue (Fig. 3). Conventional genomic DNA
extraction kits proved inadequate for the cyanobacteria. The addition of lysozyme (50
mg/mL), SDS, RNase and proteinase K in a power bead tube prior to homogenization
lyophilized the cyanobacterium and aided lysis of the L. majuscula cell. Precisely, the SDS
removed lipid polysaccharide.15 High molecular DNA (8 kb) of L. majuscula was
subsequently extracted with phenol : chloroisoamyl alcohol (25:24:1) to afford DNA with
268/280 and 280/230 ratios of between 1.8 and 2.0 and 1.7 and 2.0 respectively as
measured by spectrophotometry from the nano drop allowing for 16S rDNA identification and
19 | P a g e
complete genome sequence. The 16S rDNA of the copper sulfate exposed L. majuscula
isolates matched that of A. colombiense at 89% identity.
A 6.4 Antibiotic resistant bacteria associated with the L. majuscula and the filament
In other experiments, L. majuscula was treated to a cocktail of ampicillin, chloramphenicol
and streptomycin (4 mg mL-1 each) antibiotics in growth culture to establish drug resistant
bacteria in the consortia likely to offer protection to the cyanobacteria against bacterial
infection. It was established that P. stutzeri (yellow) S. algae (pink to orange) and K. oxytoca
(cream) resist the antibiotic cocktail treatment. The resistance towards antibiotics was
corroborated by the presence of the emrA multidrug efflux system protein emrA
[tr:G8WAU0_KLEOK], the K03543 multidrug resistance protein A and the beta lactamase
peptidoglycan glycosyltransferase gene clusters in the Klebsiella oxytoca KCTC 1686
genome.16,17 Similarly, the multidrug resistance (MDR) efflux pump F2N102_PSEU6
encoding for TbtABM operon was observed in the genome of the nitrogen fixing P. stutzeri.
Replating of detached L. majuscula filaments onto marine agar 2216 (10% w/v), identified
Pseudoalteromonas carrageenovora and Ochrobactrum anthropi as the bacteria found on
the filament. However, replating of L. majuscula specimen treated with copper sulfate
pentahydrate onto the agar did not result into any observed cultures of bacteria but instead
showed tiny specs of fragmented cells. It was also reasoned that cyanobacteria filaments left
to die would be culpable to cell wall destruction by bacteria living on the surface. To
investigate this, the filament was left to die on a microscope coverslip (48 hr) and thereafter
observed under the microscope with nigrosin stain. Nigrosin stains blue DNA and cell wall
material of bacteria and cyanobacteria. Whereas bacteria were present in a disfigured (noncylindrical) filament there were no indications of the cell wall material having been
dismembered by the bacteria during the 48 hours of decay.
20 | P a g e
A 6.5 LCMS profiling of homodolastatin 16 in L. majuscula and epibiotic bacteria
Organic extracts of freeze-dried supernatants of epibiotic bacteria (EB) were investigated for
the presence of homodolastatin 16 (1) through glass fibre (GFF 44μ) filtration and C18
purification. 2:1 dichloromethane/methanol eluants from the C-18 were evaporated down by
a rotary evaporator (23 °C) and dried with nitrogen under vacuum. Extracts were yellow in
color. Similar treatment was made for L. majuscula extracts. TD-Lyng chl was the first
Lyngbya extract fraction to elute from the C-18 column and exhibited an intense
pigmentation of chlorophyll. TD-Lyngbia eluted immediately after TD-Lyng chl. Both TDLyngbia and TD-Lyng chl fractions were observed to have similar chromatograms on the
gradient elution.
Fig. 5. Low resolution LCMS chromatograms. The times shown here differ with the
high resolution values reported in the text (Not shown here)
21 | P a g e
The molecular ion for dolastatin 15 (m/z 837.9050) consistent with the molecular formula
C45H69O9H6 was used as a standard. Dolastatin 15 was eluted after 8.56 minutes.
Homodolastatin 16 (1), antanapeptin A (2) and dolastatin 16 (3) were eluted at 12.06, 12.53
and 11.39 minutes respectively. The peak at 10.42 minutes is a contaminant from the
column unrelated to the extracts. The molecular ion m/z 915.5178 M + Na++ was consistent
with the molecular formula C48H72O10N6 for homodolastatin 16 (theoretical mass m/z
915.5202) whereas m/z 759.4290 M + Na++ corresponded with the molecular formula
C41H60N4O8 for antanapeptin A (theoretical mass m/z 759.4314) that was previously isolated
from L. majuscula along with homodolastatin 16 (1).2 The minor metabolite for the molecular
ion m/z 901.5041 M + Na++ in the chromatogram is consistent with the molecular formula
C47H70O10 for the potent anticancer agent dolastatin 16 (3) (theoretical mass m/z 901.5046)
and differs from homodolastatin 16 (1) by a methylene group.
Examination of the chromatograms and TOF MS ESI+ spectra for the epibiotic bacteria
isolates did not show any matches for homodolastatin 16 (1) and its analogue dolastatin 16
(3). Neither was there observed any signal matching with that of antanapeptin A (2).
Representative spectra for the γ-proteobacteria (Enterobacter cancerogenus, Pseudomonas
carrageenovora, Pseudomonas pseudoalkaligenes, Yorkenella regensburgeii, Klebsiella
oxytoca), Firmicutes (Staphylococcus saprophyticus), and α–proteobacteria (Ochrobactrum
anthropii) are presented here (Fig. 5). These spectra also accounted for the bacteria closely
associated with L. majuscula and those found on the filament of the cyanobacteria.
A 6.6 Bioinformatics prediction for NRPS modular compounds
The absence of homodolastatin 16 (1), anatanapeptin A (2) and dolastatin 16 (3) in the
bacteria isolates prompted investigating whether this outcome was consistent with
bioinformatics driven prediction. To ascertain if the cyclodepsipeptides found in the Kenyan L
.majuscula had templates in M. producens, NRPS adenylation scaffolds of 11 prokaryotic
22 | P a g e
microorganisms encoding the AMP-C family developed from orphan-proline genes were
blasted onto the M. producens genome.16 Long chain fatty acid-CoA ligases (5853 bp,
1951aa, 462.e-130) for PKS were established in the M. producens genome. Similar blasts on
A. colombiense resulted in long chain fatty acid but comprising fewer base pairs and amino
acids (1512 bp, 504 aa, 157.e-38) compared with the cyanobacteria. NRPS gene clusters
were found in P. putida encoding for pyoverdin siderophore biosynthesis (10413 bp, 3471
aa, 410.e-114), K. oxytoca for the siderophore enterobactin synthetase (3882 bp, 1294 aa,
840.e0) and yersiniabactin (6099 bp, 2033 aa, 233.e-61).
A 6.7 Putative biosynthesis of dolamethyleuline and dolaphenvaline fragments
Dolamethyleuline is a fragment in homodolastatin 16 (1) and dolastatin 16 (3) whereas
dolaphenvaline (Dpv) has been observed in both 1 and 3; in kulokekahilide,13 and
pitiprolamide.14 Valine in step i (Scheme 1) undergoes degradation into fatty acid
biosynthesis via isobutyryl-CoA utilising the enzymes valine dehydrogenase (EC: 1.4.1.23)
and the branched chain amino-acid aminotransferase (EC: 2.6.1.42) .
Scheme 1
The dehydrogenation in step ii involves 2-oxoisovalerate dehydrogenase E1 component,
alpha subunit (EC: 1.2.4.4) and 2-oxoisovalerate dehydrogenase E2 component
(dihydrolipoyl transacylase) (EC 2.3.1.168). These are accompanied with dihydrolipoamide
dehydrogenase (EC:1.8.1.4) for co-factor recycling and 2-oxoisovalerate ferredoxin
oxidoreductase alpha subunit (EC:1.2.7.7). The dolamethyleuline β-amino acid is afforded
23 | P a g e
through a polyketide/fatty acid extension with methyl malonyl CoA and β-transaminase in
step iv. Bacillus licheniformis, Marinobacterium stanieri, Shewanella sp. and Pseudomonas
putida isolated from the Kenyan L. majuscula putatively can synthesize the 2-oxoisovalerate
dehydrogenase E1 component, alpha subunit. The accession numbers of the respective
proteins of these bacteria are (WP_016885941.1), WP_010322356.1, WP_011622791.1,
WP_010955110.1 respectively.
Dpv is synthesized via a benzoyl-CoA biosynthesis into the phenol intermediate of a final
transaminase component by an enzyme with aldolase functionalities similar to those of
Nikkomycin B (NikB, Scheme 2 above). The NikB gene has been observed in the
Streptomyces. In this study it has been found in the genomes of Klebsiella oxytoca
(WP_004134764.1) and Pseudomonas putida (NP_745483.1). The gene cluster was absent
in the nikB genome blast of the cyanobacteria M. producens.
Scheme 2
The putative presence of the genes for the Dml and Dpv fragments in bacteria and their
absence in M. producens suggested that there could be a symbiotic relationship in bacteriaL. majuscula consortia. This led to the isolation and identification of culturable bacteria from
L. majuscula.
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A 6.8 Molecular identification of the Kenyan “Lyngbya majuscula”
The failure to identify homodolastatin 16 and antanapeptin A genes in the Moorea
producens genome raised concerns on the identity of the Kenyan marine cyanobacterium.
The Kenyan L. majuscula had only been identified morphologically consistent with other
species collected worldwide in pantropic geographical locations. Morphological identification
is limited and unreliable because of the immense diversity of the Oscillatoriales. Molecular
identification is highly accurate and specific as it is based on the genomic content of the
organism. Whereas the technique works efficiently for axenic species, molecular
identification of non-axenic cyanobacteria is especially difficult due to the presence of
bacteria and other microorganisms that complicate genomic DNA isolation. Presently the
identification of non-axenic cyanobacteria mostly utilises the multiple displacement
amplification (MDA) method which has only a limited total genomic coverage.
Various approaches for obtaining axenic cultures of cyanobacteria are well documented
including treatment of cyanobacteria cultures with toxic chemicals and mechanical
separations.17 However these methods do not elaborate on how to isolate genomic DNA
from non-axenic strains. In this study the Kenyan L. majuscula was treated to toxic copper
sulfate (CuSO4.5H2O) at different time intervals (0, 5 min, 15 min, 30 min, 60 min) with
intermittent mechanical separation prior to DNA extraction. Controls in which the toxic
chemical was not applied were used. Freeze drying of the samples in liquid nitrogen followed
by periodical thawing and sonication (30% pulsar, 10 min maximal amplitude) removed
residual bacteria.18 For obtaining genomic DNA of the cyanobacterium, homogenized L.
majuscula pellets were exhaustively extracted for bacteria genomic DNA. The resulting
bacteria DNA was of mixed species and did not therefore generate a 16S rDNA sequence.
Surprisingly, gDNA isolation of the residue largely comprising of cyanobacteria provided
quality16S rDNA sequences with 260/280 and 260/230 ratios of between 1.90 and 2.29 in
the qubit assay respectively. Whereas both controls and copper sulfate treated samples
generated 16S rDNA sequences, sequences of the latter had fewer nucleotide bases to
25 | P a g e
afford sufficient coverage for complete and/or draft genome sequence. These observations
were corroborated with degraded DNA for the copper treated samples on an electrophoresis
gel (Fig.6 below) and deformed morphologies of the L. majuscula filament (Fig. below).
Fig. 6. Lane 1: Gene ruler DNA ladder mix; Lane 2: TD01 (control); Lane 3:
TD Conv (Supernatant treated with CuSO4.5H2O); Lane 4: TD Res (Residue
treated with CuSO4.5H2O); Lane 5: Lambda DNA/Hind III marker.
Fig. 7. Lyngbya filament treated with CuSO4.5H2O for 15 minutes (left) and
for 60 minutes (right)
26 | P a g e
A 6.9 Analysis of sequence data and phylogeny of the Kenyan L. majuscula
Differential DNA isolation of the homogenised Kenyan L. majuscula commensed with a
sample treated with toxic copper sulfate, generating 16S rDNA sequences with matches for
Aminobacterium colombiense at 89% identity and 94% coverage. The control in which the
cyanobacterium was not treated with copper sulfate did not generate any sequence. We
made the assumption that there may have been cross reaction with the cyanobacteria
primers CYA 106F (CGG ACG GGT GAG TAA CGC GTG A) and CYA 781R (GAC TAC
TGG GGT ATC TAA TCC CAT T) during amplification. Mismatches arising from the primer
CYA 106F are not unusual.19 Arguably the low % identity was questionable. Furthermore,
only small concentrations of the DNA were obtained on isolation. Treatment with copper
sulfate of a cyanobacterium residue rather than the usual supernatant of a TE buffered
solution resulted into a 16S rDNA sequence matching with that of Cylindrospermum stagnale
at 85% identity of 100% coverage and 88% at 95% coverage respectively for different
aliquots.
With this uncertainty the method was tested against the known L. majuscula CCAP 1446/4
strain from the Culture Collection at Oban, Scotland. Both the supernatant and the residue
confirmed the identity of the strain with 100% identity for 100% coverage of 6 replicates. Still
a major drawback was to get a non-degraded DNA with the quality suited for draft and/or
complete genome sequence. The isolation of soil bacteria DNA assumes exhaustive
extraction of bacteria DNA with the residual soil and humic substances as substrate for the
bacteria. In an analogy with this study cyanobacteria residue was the substrate comprising
the bulk of the cyanobacteria genomic DNA material. Exhaustive extraction of bacteria
genomic DNA from the Kenyan L. majuscula followed by genomic DNA isolation of the
residue with copper sulfate and controls respectively generated 16S rDNA sequences with
sufficient nucleotides for a blast. Whereas the copper sulfate treated DNA was degraded as
observed in an electrophoresis gel (Fig. 6 Lanes 3 and 4 respectively), the control (Fig. 6
Lane 2) was not and was of good quality to generate an assembly library for a draft genome.
27 | P a g e
An NCBI blast of the generated sequence of the Kenyan marine cyanobacteria, without
restricting organism identity matched the sequence at 99% identity with an uncultured
Aminanaerobia bacterium. 16S rDNA fragments of this organism had up to 100% identity
match with the aforesaid uncultured Aminanaerobia bacterium. These observations were
consistent with all the 16S rDNA sequences obtained from the CuSO4.5H20 extractions at 0,
5 min, 10 min and 30 min. CuSO4.5H20 was found to fragment the cyanobacteria genomic
DNA. With the blast restriction to cyanobacteria all the sequences were matched with an
uncultured cyanobacterium respectively. 16S rDNA sequences obtained from the axenic
Lyngbya majuscula strain CCAP 1446/4 by the aforesaid method did not show any matches
to Aminanaerobia bacteria but instead consistently matched with 100% to L. majuscula. A
phylogeny carried out for the Kenyan “Lyngbya majuscula” showed quite distant relations
with L. majuscula CCAP 1446/4 and its clones.
The goal of the project was to identify gene clusters encoding for the anticancer
homodolastatin 16, antanapeptin A and the potent anticancer dolastatin 16 originally isolated
from a Papua New Guinea sea hare; and to carry out the expression and recombinant
biosynthesis of the anticancer compounds in a heterologous Saccharomyces cerevisiae
system. Work on the draft genome of the Kenyan marine cyanobacterium is ongoing at the
University of Aberystwyth in collaboration with Dr Justin Pachebat. This expected draft
genome of the Kenyan cyanobacteria shall reveal the true identity and nature of the
organism.
A7
Manuscripts
On manuscript preparation, we are shortly due to submit the manuscript ‘Bacteria living on
marine cyanobacteria utilise biofilm exopolysaccharides desiccation and avoidance to resist
UV irradiance’ to Photochemistry and photobiology C journal of Japan. Currently the
manuscript is on the proof reading stage. I have also drafted a manuscript “Differential DNA
isolation as a novel method for identifying non-axenic cyanobacteria” based on a novel
28 | P a g e
technique that is likely to replace the multiple displacement amplification currently used to
obtain the draft genome of non-axenic cyanobacteria. A key feature of the publication shall
be the observation that molecular identification of the Kenyan L. majuscula is not consistent
with the morphological identification previously done by Mirjam Girt of the Oregon State
University in 2003. The manuscript is for submission to the Proceedings of the National
Academy of Sciences (PNAS) journal of the USA for publication.
Separately we shall soon publish the draft genome of the Kenyan “Lyngbya majuscula” in
the Journal of Microbiology. Additionally, our confirmatory LC/MS results and draft genome
data shall strengthen our resolve to publish our findings on the source of the anticancer
homodolastatin 16, dolastin 16 and antanapeptin A in Nature biotechnology.
B
IMPACT
B1
Seminars and guest lectures
In regard to seminars and seminars, I provided the ‘Cyanobacteria-bacteria interactions’
lecture for the MST3011 Marine Microbiology Mini-Module at Newcastle University in 2012
and “The discovery of novel pharmaceutically relevant natural products from marine
cyanobacteria”; and “The future prospects for biodegradable resins from marine
cyanobacteria” for the 2013 Marine Biology group. Feedback from the Marine Biology
Research students was quite good at an average 9/10. I also presented a talk entitled
‘Biosynthesis of the anticancer cyclohexadepsipeptide homodolastatin 16’ to Professor Ian
Head’ research group at Newcastle University and externally I was invited to give a talk on
the isolation and biosynthesis of the modular anticancer cyclohexadepsipetide to the
Research Group of Professor Rebecca Goss at University of St Andrews, Scotland, UK in
April 2013. On outreach I was a guest speaker at the EU Marie Curie conference held at
Durham University in May 2013, a seminar that was organized for North East England.
29 | P a g e
B2
International conferences and symposia
B 2.1 Federation of European Biochemical Society (FEBS) 2013 conference at St
Petersberg, Russia from 6-12 July 2013.
With regard to international conferences, I was invited to present my talk “Biosynthesis of the
modular anticancer cyclohexadepsipeptide homodolastatin 16” at the Federation of
European Biochemical Society (FEBS) 2013 conference at St Petersberg, Russia from 6-12
July 2013. This was an especially prestigious ‘Mechanisms in Biology’ conference in which
11 Nobel laureates comprising 7 in Chemistry and 4 in Medicine or Physiology attended.
They included:
1. Sidney Altman (USA) who won the Nobel Prize in Chemistry 1989 “for the discovery
of catalytic properties of RNA” together with Nobel laureate Thomas Cech.
2. Nobel laureate Aaron Ciechanover (Israel) who was awarded the Nobel Prize in
Chemistry in 2004 “for the discovery of ubiquitin-mediated protein degradation”
together with Nobel laureates Avram Hershko and Irwin Rose. Nobel laureate Jules
Hoffman (France) who won the Nobel Prize in Medicine or Physiology together with
Nobel laureates Bruce Beutler and Ralph Steinman in 2011 “for their discovery
concerning the activation of innate immunity”.
3. Nobel laureate Robert Huber (Germany) who together with Johann Deisenhofer and
Hartmut Michel were awarded the Nobel Prize in Chemistry in 1988 “for the
determination of the three-dimensional structure of a photosynthetic reaction centre”.
4. Nobel laureate Roger Kornberg (USA) who was awarded the Nobel Prize in
Chemistry in 2006 “for his studies of the molecular basis of eukaryotic transcription”.
5. Nobel laureate Jean-Marie Lehn (France) who together with Donald Cram and
Charles Pedersen won the Nobel Prize in Chemistry in 1987 “for their development
and use of molecules with structure-specific interactions of high selectivity”.
6. Nobel laureate Richard Roberts (UK) who together with Phillip Sharp were awarded
the Nobel Prize in Medicine or Physiology in 1993 “for their discovery of split genes”.
30 | P a g e
7. Nobel laureate Jack Szostak (USA) who together with Elizabeth Blackburn and Carol
Greider were awarded the Nobel Prize in Medicine or Physiology in 2009 “for the
discovery of how chromosomes are protected by telomeres and the enzyme
telomerase”.
8. Nobel laureate Susumu Tonegawa (Japan) who won the Nobel Prize in Medicine or
Physiology “for his discovery of the genetic principle for generation of antibody
diversity”.
9. Nobel laureate Kurt Wuethrich (Switzerland, USA) that was awarded the Nobel Prize
in Chemistry together with John Fenn and Koichi Tanaka in 2002 “for his
development of magnetic resonance spectroscopy for determining the threedimensional structure of biological macromolecules in solution”
10. Ada Yonath (Israel) who in 2009 was awarded the Nobel Prize in Chemistry together
with Venkatraman Ramakrishnan and Thomas Steitz “for studies of the structure and
function of the ribosme”
I attended nearly all the plenary sessions by the Nobel laureates and experienced their
humility in servitude to science for humanity’s sake. In all their sessions it became clear that
their approach is towards focusing on a problem “the goal” rather than allegiance to a
discipline of science. This tremendously influenced my research during the last half of my
Marie Curie Fellowship at Newcastle University. There was also much furor regarding taking
photographs with Nobel laureates at the conference especially from our British
conservatives. However, I argued that not a single African was honored with the award of a
Nobel Prize in Chemistry and Medicine or Physiology and was therefore allowed the
privilege. Subsequently I felt most humbled and yet honored to freely interact with Nobel
laureates Jack Szostak, Jules Hoffman and Susumo Tonegawa.
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B 2.2 International Advanced Single Cell Biotechnology at Sheffield University, UK
on 12 February 2014
I also attended the “International Advanced Single Cell Biotechnology” at Sheffield
University, UK on 12 February 2014. The one day symposium was hosted by Dr Wei Huang
of the Kroto Research Institute. The key note speaker was Professor Michael Wagner of the
University of Vienna. There were presentations from all over the UK including Imperial
College, London; Sanger Institute; Manchester University and there were also presentations
from the USA. I was mostly interested in the symposium because of the difficulties I had
encountered in isolating quality DNA from the Kenyan L. majuscula for complete/draft
genome sequencing. The meeting was especially useful because I learnt some techniques
that were most helpful towards my research. There were also a number of questions
remaining unanswered in my project. I did find colleagues to partner with for my future
research aspirations. In this regard I single out a project which aims “to investigate the role
of cyanobacteria toxins on bacteria cell division and cell modulation and the relevance of
cyclodepsipetides in cancer therapy”. It was a happy and exciting moment to realize that Dr
Huabing Yin of Glasgow University had almost similar interests to mine albeit unconsciously
and could not help hugging her after the end of her seminar. The symposia aided my
networking and besides my on-going work on single cell technology at the University of
Aberystwyth Wales, UK, prospects for working with Dr Wei on Raman Tweezer
spectroscopy and with Dr Yin are high. It also afforded me the opportunity to realize that the
UK is a giant in science, something I always took for granted.
B3
Outstanding outcomes of the project
B 3.1 Grant Applications
Regarding achievements and outstanding milestones, the isolation of bacteria pathogens
from a marine cyanobacterium opened the window to relooking into the biogenesis of human
pathogenic toxins in portable water from Kenya, Tanzania and South Africa and in this
regard we submitted a £1.24M proposal ‘Understanding the source of microbial
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contamination in African coastal borehole waters’ to the Royal Society DFID Capacity
Building Initiative in April 2014. This intended project shall dissect the chemistry and biology
of bacteria and protozoa pathogens in coastal borehole water, aiming to finding solutions to
waterborne diseases in Africa and worldwide. The project shall link scientists in the UK from
Newcastle University, Aberystwyth University, St. Andrews University with those from Pwani
University, Kenya; University of Dar es salaam, Tanzania and the University of Cape Town,
South Africa. We are awaiting the outcome of this application due in October 2014.
Earlier on a scholarship was awarded to a summer student at the School of Chemistry by
Newcastle University to investigate the synthesis of some fragments in homodolastatin 16
with the aim of tracing its origin and whether or not the fragments originate from the
cyanobacteria or EB or both. This project was in collaboration with Dr Michael Hall of the
School of Chemistry who advised me on Chemistry related issues of the EU Marie Curie
project.
B 3.2 Outreach program - Mentoring of Marine Biology students
The EU Marie Curie Fellowship generated two projects for Marine Biology Honours students
at Newcastle University namely; “The role of secondary metabolites in Bacillus licheniformis
UV-resistance” and “Exploring Marine Bacteria Polysaccharides from a Desiccated
Environment and Evaluating their Hygroscopic Abilities in Application to the Cosmetic
Industry”. I supervised both projects and have co-authored a manuscript for publication in a
peer reviewed journal along with my own research findings on UV-resistance with the
students. Subsequently, these students are considering pursuing PhD studies in the UK.
Additionally, nine undergraduate students undertook their projects with our research group
during my fellowship as a result of the lectures I gave to them on cyanobacteria-bacteria
interactions and on marine biotechnology and drug discovery.
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C
THE UNITED KINGDOM AND MY EU FELLOWSHIP
I very much enjoyed the experience of being an EU Marie Curie IIF Research Fellow in the
United Kingdom. The independence of thought and the resolve to make a contribution
towards research in the EU was a strong motivation for my work. It was evident that my stay
had impacted on me positively; and made a lot of friends in addition to embracing the dry
humour of the British people. Unfortunately non EU Marie Curie citizens are taxed heavily on
money which does not originate in the UK when in reality they do not enjoy the same
privileges as locals. Nevertheless, I would still recommend the UK as a destination for early
career scientists to develop their expertise.
D
ACKNOWLEDGEMENT
I wish to thank Professor Grant J Burgess for hosting me at the School of Marine Science
and Technology and for his enduring support; Dr Michael Hall of the School of Chemistry for
helpful discussions and mentorship during my project. Jill Cowans at the Dove Marine
Laboratory arranged my purchases for consumables. I wish to most sincerely thank Ms Lisa
Inganni and Anthony Gibson for handling my finances. Lastly, I acknowledge the EU for
according me the opportunity to work as a Marie Curie IIF in the UK through their funding.
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8. Pettit, G. R., Smith, T. H., Xu, J., Herald, D. 2011 New crystal dolastatin 16 having
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Annex 1. Table 1 of Kenyan Lyngbya majuscula epibiotic bacteria (EB) isolates
Accession Strain
Taxon
Shewanella algae
KC660130
SHALG-01
99
γ-proteobacteria
Shewanella algae
KC660131
SHALG-02
99
γ-proteobacteria
Marinobacterium stanieri
KC660132
MARIS-01
99
γ-proteobacteria
Acinetobacter johnsonii
KC660133
ACJ-01
99
γ-proteobacteria
Marinobacterium stanieri
KC660134
MARIS-02
99
γ-proteobacteria
Staphylococcus saprophyticus
KC660135
STAPRO
99
Firmicutes
Pseudomonas stutzeri
KC660136
PST-01
99
γ-proteobacteria
Enterobacter cloacae
KC660137
ENTCLO
99
γ-proteobacteria
Cellulosimicrobium cellulans
KC660138
CCL-01
99
Actinobacteria
Cellulosimicrobium cellulans
KC660139
CCL-02
99
Actinobacteria
Pseudomonas pseudoalcaligenes
KC660140
PPS
99
γ-proteobacteria
Pseudomonas putida
KC660141
PPT
99
γ-proteobacteria
Bacillus aereus
ND
ND
99
Firmicutes
Bacillus licheniformis
KC660142
BLC-01
99
Firmicutes
Bacillus licheniformis
KC660143
BLC-02
99
Firmicutes
Bacillus subtilis
KC660144
BS-00
99
Firmicutes
Pseudomonas stutzeri
KC660145
PST-02
99
γ-proteobacteria
Enterobacter cancerogenus
ND
ND
99.24
γ-proteobacteria
Klebsiella oxytoca
ND
ND
99.23
γ-proteobacteria
Yokenella regensburgei
ND
ND
99.02
γ-proteobacteria
Ochrobactrum anthropic
ND
ND
99.88
α-proteobacteria
Pseudomonas stutzeri
ND
ND
99.87
γ-proteobacteria
Pseudoalteromonas
ND
ND
99.22
γ-proteobacteria
carrageenovora
ND – Strain sequences not deposited with Genbank but were inferred from Blast
37 | P a g e
Annex 2. Phylogeny of EB isolates
38 | P a g e
Annex 3: Adenylation domain for Moorea producens
>gi|332705439|ref|ZP_08425517.1|/1-2887 amino acid adenylation domain protein [Moorea
producens 3L]
-----------------MNLSEFLQELVISGWQFWA----EEGQVCFQAPDADSTDQVLAQLKQHKRDILT
ILQEHPE---VLQVYPLGYGQ-------------------------------------------------QG
IWFLWQLFPDNPNYNVSFATRIY--------SQVNVTTW---------------QQTFEALRKRHPLLCS--TFPKCGETPIRQHSEQLD-------FVQIDASTWDENELQTQVVAAHRHPFDLQTDPVMRVRWFTRSEQE
-------HILLLTIHHIAWDGSSANI------IVKELS----ELYQAHCAGVAVDLPSLQHT---------YQDYVKWQ--------QQLVEGSKG------ESLWTYWQQQLAGELPVLNLPTDRPHPPIQTNNGAVYRFQ
LPEHLVTQVKALSQAEGATLYMTLLAAF-------------QVLLHRYTG-------QEDILVGSPTSGRT-RPEFTSVVGYFVDSMVMRAKVSGSLSFREFLTQVRQ-------TVIDALAHQDYPFSLLVEKLQP--------------ERDLSRSPIFQVF-FGLHNFLQSETQQLFLGETKTLVHWGGMEVETFLFDQYESLEDLVL-----------EIIEINSQLSGFFKYNTDLFDEQTIAQMASHLQTLLAGIVT-----------HPEQRLESL
P------LLTQAEQHQLLVEWNQ--------------TTTHYPTDKCIHQLFEEQVEQTPDAI--------A
VVFKEEKLSYQELNIRANQLARYLQSLGVSPEV-LVGVC--------------VERSLEMIVGLLGILKAG
GVYVPLDPKYPQ-------------ERLDYMFRD--SQMSVLLTQQQLLTLLPQYEAK-----------------VVCLDRDWQKIVTEN-----------------------------PKNVTSEVTAENLAYVIYTSGSTG
KPKGVMVAHIGLHNLLKVQIQAFKVSSNSRVLQFASLSFDASIWEIVMALGSGASLY-----LESRENLL------------------PGASLSKWLNEKKITHLTLPPSALAVM-------QKEELPSLQTIVVAGEACPA
EVISQWSQGR---QFVNAYGPTESTV-----CATMAE------CSPEYSVLP---IGHPIANTQI----YLL
DNNLQP--VPIGIPAEMYIGGIGLARGYLN----------------------RPDLTTQKFIPNP-----FS
NKAEQRL----------------YKTGDLARYLPDGNIEFLGRI--------DHQVKIR----GFRIETAEI
EAVLNQNPTVKQTVVVA-REDKPGDKHL-------------------CAYIVAQMETATNSNPE--LSETHL
NSW-QEIFNQQIYSQ--LSEVTDPLFNTTGYLSNYDKQP--IPEAQMRDWAEDIVTQV-------LANKPN
SVWEVGCGTGMLLFKIAPHTRAY---------------------------YGTDISEVSLKYIQTQIAQQPD
KYAHVTLAQKAAEEMADIADNSFDVV-------LLSS-----IVQYFPSVEYLLQVI------SNSIRVVKP
GGMIFLGDIRSL--PLMRAFHTSVQLHKAPPSLSVQQLKQGIY---RLMQQETELLVSPEL-FVALKDTYP
--EITHVQI-RLQRG----SEHNELNKYRYSV-LLHIQAKPTSVIVAPVENGVGMSMEDIEVYLGQQQPES
ICFSSL-------TNGRVATDMAAVELLSQVESKLNVQQLRQQLRQKLVNGIEPEQLH--QLSASLGYELE
LC------------WSHKTEGCFDAVFVRSSLAPEAM-VLTPLTQQSVVGGNWHRYGNNPLASVTGKQLIP
Q-WR------------KYLEERLPEY-----MVPSRYVILP-QLPLTPNGKV-----------NRKAL---------------------------------------PAPDNTSSRSTEFVAPETSTEKAL--AAIWAEVLSI
-----QQVGIHDNFFESGGHSLLATQVVSRIRQALGKELTLQRLLESPTIAELDSALVQLPRVEDSP
KQKP
DGLLPTIVPAPSQRYQPFPLTEIQQAYWLGRNSHFDLGNITTHGYLELDCENLALDRLSQAW
QQVIDHHDML
RMVILPNGEQQVLEQVYPYQIEVLDLRGQPEQIVSTELETIRYRLSHEMFPAGEWPLFKIRV
TRLADQRYRL
HWSFDALIADAWSMIIVWQQWLQLYQNPDSFLPKLDLTFRDYVLAELSLKDTPQYRRSQQY
WWNRLETLPPA
39 | P a g e
PELPLVKQTATLEQPEFNCYRAELSAPDWQQLQARAKQASLTPSGVLLAAFADFLAYWSKS
PKFTINLTLFN
RLPLHPQVNDLVGDFTSLTLLEVNQKNAAPFAQRAQRLQGQLWQDLDHRYVGGVEVQRE
L-RRQRGSYQPMG
VVFTSTLALNTSAEKGLPSNEWHAWPFDQLGETVYMVSKTPQVWLDNSVAEQNGALLLIW
NVVEDLFPEGFL
NDMFTSYYHWLQQLATSDVAWAQTCPQLLPLSQLTQRLQVNETYAPVSEETLHNLFVKQV
QQRPEAIALITP
QRTLTYHELYTEAQALGQQVQQLGATPNTLVAVLMEKGWEQIVAVLGILMAGAAYLPIDAAL
PQERQWSLLE
QGEVKLVVTQAALNASLGLPDHLHCLVVASQPQEIIDTPLEANVSSSDLAYVIFTSGSTGTPKGVMIDHRG
AVNTIQDINQRFDVQPTDRMLAVSALNFDLSVYDIFGLLAAGGTLVMPTPEAAKDPVHWVEL
MTTHQVTLWN
TVPALMQMLVEYLSEHPDQVTEDLRLALLSGDWIPLNLPTQIQSLWPQGQVVSLGGATEAS
IWSVYYPITTV
EPEWKSIPYGKPLVNQSLHVLNHNLDPCPNWVPGQLYIGGIGLAQGYWRDEQKTNASFILH
PQTGERLYKTG
DLARYLPDGNSEFLGREDFQVKISGYRIELGEIEATLLGHATVKETVVAAVGELQSKQLVAYVVFHSESSSDSATEDVHD-------------DMRIDELRHYLQQQLPEYMVPPSYMVLDALPLTANGKVDRKRLPTP-ELISDHYSPDTYIPPRNHQELQLVKLWEEILEVQPIGVGSHFFDLGGHSLLAVRLMNRIEQDF
GRSLPLATL
FQAPTIEQLAVILQQEQGVPTLSPLVPIQTQGNQPPIFCVHPAGGTVFCYLELSQLLGANQP
FYGLQSLGQQ
EGQAPLTTVEEMANVYLAAIREVQPQGPYLLMGWSFGGMVALQMAHDLLSQGEQVAFLGL
LDTYAPAHMPDE
QVLSEDVEVLLELFGGPLSLDWEVLRDLPSEQQSALIWEQAHQANLVPPDLGAAQIERLLQ
LMKLNHKAMRS
YSPPDYPDVITLLHAEAGSVAVSSTEVTTDPTLGWQAISPSKVEVHTIPGYHEYMVYQPTVV
IVAETIKADI
EKGLNTDVETSSK
>gi|332712440|ref|ZP_08432366.1|/1-3195 amino acid adenylation domain protein [Moorea
producens 3L]
-----------------MAELNLNRDLGTSNSEVVQLTELGNGVVQITMKDESSRNGFSPSIVEGLRHCFS
VVAQNQQ--YKVVILTGYGNYFSSGASKEYLIRKTRGEVEVLDLSGLILDCEIPIIAAMQGHSFGGGLLLG
LYADFVVFSQESVYATNFMKYGF--------TPVGATSLILREKLGSELAQ--EMIYTGENYRGKELAERG
IPFPVVSRQDVLNYAQQLGQKIAKSPRLSLVALKQHLSADIKAKFPEAIKKELEIHQVTFNQP
EIASRIQQE
FGETVIPNLIQSTVEQKIPNPQPVQLRIPSYGLLKNLTWMPQERRKPKSTEVEVQIKAVPVNF
REVLNVLGI
FQEYIKKRYRSGIISAENLTFGVEGVGTVVAVGSDVSQWK---VGDEVILAYP--------GNAFSSFVIC
SPDDLLAKPSDLSMVEAATIFMSFFTAYYGLHNLAKVQPGERVLIHAASGGAGQAAVQLAQ
FFGSEVFATT-SPHKISVLREQGIKHVMNSRTT------EFASEVRELTQGNGVDVIFNSLTHGEYIPKNIDILAPGGRYI
EIGRLNIWSHEQVSQRRPDVKYFPFDMSDEFVRDKQFHAKLWDDLALLFESGSLKPLPYKVFPS-EDVVEA
40 | P a g e
FRHLQHSKHIGKIVVTMPELYNGVKNSSQQANQESMSHQEELLHQLQSGDISLENAEQLLL
GLTDQQILATV
PNNGQNKLINTDKTEQILSLLSSGEISLENAQNLLETVDLNSPTKKNLPTAVPNQGQSNQDE
AILNQLQSGE
VSLEDAEQLLLEIQQKESVTTKSIPDQRITDDIAIIGISCRYPGAKNWKEFWENLKHGVDSVT
EPPPGRWEG
RSWYHSDPEHPGTACSKYAAFLDDIDKFDPLFFQISPGEAELIEPQQRIFLEEAYHAIEDAGY
APDSLKGKH
CGVFVGAASSDYIKFLSNSGFGHHRLVLSGTMLSVLPARIAYFLDLKGPVVAVEAACSSSLV
AVHQACESIK
RGESEIAIAGGISTMLTPDFQVLSSQFQMVSPEGRCKSFDAEASGIVWGEGCGAILLKRYEQ
AVQDQDHIYG
IIKGTGTNYDGSTNGISAPSSKSQARLAENIYQQFGINPETISYL------EAHGTATPLGDPIEVEAF-T
EAFSKWTAQK---QFCAIGSVKTNIGNAATAAGMSSLIKTILCLKNQKLVPSLHFNQPNPNIDFANSPFYV
NTEFKAWEVPTGIPRRAAVNSFGLNGTNAHVVVEEAPIEDNRQTSPVSPQGGKATGNSED
YLENSVHLLTLS
AKTETALGEVISSYQNYLKTNPNLRLGDVCYTASTGRTHFTHRLAVVAPNQQELVEKLRQH
QEGKKLAGITS
GELLNNTTVAKIAFLFT-GQ--GSQYINMGKQLYQQAPTFRQAINQCEEILSSVETFQETSLRNILYPTDK
NSSGSSLLGQTAYTQPALFAIEYALFK--LWQSWGIEPDVVMGHSVGEYVAATVAGVFSLEDGLKLIAARG
SLMQKLPGDGKMLWAMAPESKVLETLKAKDLSEKVAIAAINGPQSIVISGEGKAVEAIATNLE
SAGITTKPL
KVSHAFHSPLMEPMLAEFEAAAKEITYEQPRIPLISNVTGKQVTEQITTAEYWVNHVRQPVQ
FAQSMKTLYQ
EGYELFLEIGPK--PVLLSMGRQCLPEKI-GVWLPSLRPGVEECQQMLSSLGKLYVEGAKVDWIAFEQNYA
RQKVALPTY-PFQRERYWVSSQNGYEQKSY----WLKGKEQHPLLGEKINLAGIEDQHRFQSYIGAESPGY
LNHHQVFGKVLFPSTGYLEIAASAGKSLFTSQEQVVVSDVDILQSLVIPETEIKTVQTVVSFAENNSYKFE
IFSPSEGENQQTPQWVLHAQGKIYTEPTRNSQAKIDLEKYQAECSQAIEIEEHYREYRSKGI
DYGSSFQGIK
QLWKGQGKALGEMAFPEELTAQLADYQLHPALLDAAFQIVSYAIPHTETDKIYLPVGVEKFK
LYRQTISQVW
AIAEIRQTNLTANIFLVDNQGTVLVELEGLRVKVTEPVLTQKSAFKEQLKSASVSERQELLTT
QISSAIVNI
LGLRDGQQIERHQPLFDLGLDSLMAVELKNQLESNLGTSFSSTLLFDYPTVESLVEYLANNV
IPIDSFSE---LPTLIPHPEQRYQPFPLNDIQQAYWIGRNQIFDLGNIATHIYIEVDCENLNLESLHQAWRRLID
HHDML
RMVVLADGNQQILEQVPPYEIEILNLSEESPETIASELEQIRNQMSHEVLPTNQWPLFHLRAT
RLNEQCFRL
HASIDMLIFDAWSTYVLFKQWSELYNNPQSSLPATEISFRDYVLAELELKDSPQYLSSQQYW
FNRLDNLPPA
PEIPQAKVTSAITDPQFNTHTAQLSQSDWQQLKNKASKANLTPSGVLLSAFASVLNYWSKS
SKFTLNLTLFN
RLPLHPQVNELIGDFTSVILLEVDNSQAVPFISRAQKLQRQLWEDLEHRYISGVEVQRELYR
R--GRSQPMG
VVFTSTLGLKSLADEEVG---RGFGLEHFGEVVYSAAQTPQVLLDHIVTEEKGALAFSWHTVEGLFPEGLI
41 | P a g e
EQMFEAYCDLLQQLATSDEPWMETYHQLLPTAQLALQAQVNQTTQSWSEDILHSLFVKQV
QVQSEATAVISP
QKSLTYGELYQRSHQLGHGLRKLGVKPNQLVAVVMEKGWEQVVAVLGILMSGGAYLPIDP
GLPQERQWYLLE
QAQVTQVLTQTHLKQSLGWPEGIKCWSVDTEELAEYDPNPLEPVQTSEDLAYVIYTSGSTG
LPKGVMIDHRG
AINTILDINQRFKVTPSDRVLALAALNFDLSVYDIFGVLGAGGAIVMPPPKAAKDPACWRELII
AHEVTLWN
SVPALMQMLVEHLLGTSATAVGDLRVVMLSGDWLPVDLPSKIQSLWSNVQVMSLGGATEA
SIWSIGYPIEKV
GSDWKSIPYGKPLLNQSFYVLNELMEPRPVWVPGQLYIGGVGLAKGYWKNEHKTQASFIT
HPVTQEPLYKTG
DLGRYLPDGNIEFLGREDFQVKINGYRVELGEIEVALKQFPGIKEAIVTAIGESQQSKRLVAY
AVFKEKSVI
SDSSLTDIHQTEDKNEVGQPDQEINCTSEQLRKYLWQKLPEYMVPDDYVILEALPLTANGK
VDRKRLPKPQR
QTIADT--NQNILPQTKTEQQIAAVWTEVLELEEVGIHDNFFAIGGNSLLVIRVHNKLQELLGIELKVVDL
FANPTVHFLSQHLTQ-----------------------------------------IGSKELF---------------------------METSKTRG-------------------------------------------DE
RV-----------------------------------------------KKGTTRKER----------------------------------------------------------------------------------RNI
RKSLR-----GKK
>gi|375260917|ref|YP_005020087.1|/1-2032 yersiniabactin synthetase, HMWP2 component
[Klebsiella oxytoca KCTC 1686]
MISGAPSQDPLLSDNGEAADYQQLRELLIQELNVAPQQLQEESNLIQAGLDSIRLMRWLHW
FRKKGYRLTLR
ELYAAPTLAAWRQLMRSRSGEKPDDASSPAE-----------------------------------------AAWPVMSEGTPFPLTPVQHAYLTGRMPGQTLGGVGCHLYQEFAGHYLTAPKLEQAITILLQ
RHPMLHI--AFRADGQQVWLPQPYWNG--------VTVHDLRQTDEASRQAYLETLRQRLSHRLLRVEMGETFDFQLTL
LPDNC--HRLHVNIDLLIMDASSFTL------FFDELN--------ALLAGESLPPGDPRYD---------FRAYLLHQQKIN----QPLLDKARA-----------YWLAKASMLPPAPVLPLACEPATLREVRNTRRRMI
VPTTRWNAFSQRAGENGVTPTMALATCF-------------AAVLGRWGG------LTRLLLNITLFDRQP
LHPAVDEMLADFTNILLLDTACDG-----DTVSNLARKNQL---TFTEDWEHRHWSGVELLRELK---------------RQQSHPHGAPVVFTSNLGR-SLYSSRPESPLGEPE----WGISQTPQVWIDHLAFEHRGEV
WLQWDSNDALFPPALVETLFNAYCQLINQLCDDESA---------------------------WKKPFADRM
P----------QSQREIRQRVNA--------------TDAPVP-QGLLHEGIFRIALRQPQAL--------A
VTDAHYQWNYRELTENARRCAGRLIACGVQPGD-NVAIT--------------MSKGAGQLVAVLAVLLSG
AVYVPVSLDQPA-------------ARRGKIYAD----------ANVRLVLTCQHDASAWSDDIP--------------HLTWQQAIEAE-----------------------------PLADQAAHAPTQPAYIIYTSGSTG
TPKGVVISHRAALNTCCDINSRYQVGPGDRVLALSALHFDLSVYDIFGVLSAGGSLV---IVMENQRR-------------------DPRAWCELIQRHQVTLWNSVPALFDMLLTWCEGFADAAPEKLRAVMLSGDWIGL
DLPARYHAFRPQGQFIAMGGATEASI--WSNACEINR------VPDHWRAIP---YGFPLANQR----YRV
42 | P a g e
VDELGR-DCPDWVPGELWIGGIGVAEGYFN----------------------DPVRSEQQFVTQS---------NARW----------------YRTGDLGCYWPDGTLEFLGRR--------DKQVKVG----GYRIELGEI
ESALSQLAGVKQSTVVAIGE---KEKTL-------------------AAWVVPQGSAFCVTHHR---DPALP
QAW-RGLAGTLPCC----------------------VCPPEISAGQVADFLQHRLLKL-----------KPG
QTPGADPLPLMNALAIQPRWRA--------------------------------VVERWLAFLVTQQRLQPA
AEGYQVCAGE-APENDPPSFSGHDLT---------------LTQILRGARHELSLLNDARWSPESLAFDHP
ASALYIEELATICQQLSRRLQRPVRLLEV-----------GVRTARAAECLLTRL-SADEIEYVGLEHSQE
LLLSARQRLAPWSDARLALWSADTLTAHAHSADIIWLNNALHRLL---------------------PEEPGL
L--------------AALQQLAVPGALLYVLEFRQLTPSA--LLSTLLLTDGQPEAL------------------------------LHNSADWGAIFTAAAF----------NCQHGDEVEGLQRFLVQCPVSQVRRDPRQL
Q---------------SALAERLPGW-----MVPKRIFLLD-ALPLTANGKI-----------DYQTL--------------------------------------KRCHTPEAENRTEADLPLGDIEKQV--AVIWQPLLSM
------GAVSRETDFFQHGGDSLLATRLIGQLHQAGYEARLSDLFNHPRLADFAATLRKTDLPVEQP--------FVHSPEERYRPFALTDVQQAYLVGRQPGFALGGVGSHFFVEFEIADLDIHRLEKVWNRLIAR
HDML
RAVV-RDGQQRVLEQTPPWVIPA-HILHSPEEAL----QVRDRLAHQVLNPEVWPVFDLQVGFVDGMPARL
WLCLDNLLLDGLSMQILLSELEHGYRYPQQLPPPLPVTFRDYLQQPALRTPNPDSLA--WWQTQLDDIPPA
PALPLRCLPQDVETPRFARLYGAMDSARWRRLKQRAADAHLTPSAVLLSVWSTVLAAWSA
QPDFTLNLTLFD
RRPLHPQINQILGDFTSLMLLSWHPGES-WLQSARLLQQRLSESLNHRDVSAIRVMRQLARRQNVPAVPMP
VVFTSALGFEQD---------NFLARRNLLKPVWGISQTPQVWLDHQVYESEGELRFNWDFVAALFPDGQV
ERQFAQYCALLNRMAEDDSSWQ------LPLADLVPPLKVTER--------------RARRLRPERA--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------QPRIAAD-------------------------------------------------------------------------------------------------------------------------------------------KSSVSLIC-------------------------------------------------------DTFREVVGE---------------------------------------------------------------------------------------------------------------------------PVAPAENFFEAGATSLNLVQLHVLLQRHEFATLTLLDL
FTHPSPVALANYLAG-----------------------------------------VALKEK-------------------------------------------------------------------------------TK
RV------------------------------------------------------------------------------------------------------------------------------------------RPV
RRRQR------RI
43 | P a g e
Annex 4:
MODULAR
ASSEMBLY
DLYNLSLI
DAWTVAAV
MtaD-M1-Cys and TycA-M1-D-/L-Phe Biosynthesis
DOMAIN
NRPS EXPRESSED
E-BIT VALUE
MtaD-M1-Cy
gi|6635397|gb|AAF19812.1|MtaDM1-Cys|Myxothiazol synthetase
gi|6724259|gb|AAF26925.1|EpoPM1-Cys|Epothilone synthetase
gi|2982194|gb|AAC06346.1|BacAM2-Cys|Bacitracin synthetase
gi|408802|gb|AAA27636.1|Irp2M1-Cys|Yersiniabactin synthetase
18e -0.044
gi|48323|emb|CAA78044.1|AngRM1-Cys|Anguibactin synthetase
17e -0.077
gi|2623771|gb|AAC45928.1|TycAM1-D-/L-Phe|tyrocidine synthetase
1
gi|39369|emb|CAA33603.1|GrsAM1-D-/L-Phe|Gramicidin
synthetase A
gi|2623772|gb|AAC45929.1|TycBM3-D-/L-Phe/Trp|tyrocidine
synthet...
gi|2982196|gb|AAC06348.1|BacCM2-Phe|bacitracin synthetase 3
gi|440169|emb|CAA82227.1|CssAM9-Val|cyclosporine synthetase
15 0.38
18e -0.065
TycA-M1-D-/LPhe
18e -0.044
18e -0.044
17e -0.077
18e -0.065
17e 0.10
16e -0.16
15e -0.38
HYPOTHETICAL None
N/A
N/A
PROTEIN
10623 amino acids comprising 38 domains were found in the blast for M. producens. 4 of the
domains were for adenylation with 2 encoding MtaD-M1-Cy, 1 TycA-M1-D-/L-Phe and 1
hypothetical protein. The E-BIT values are exp (-ve).
44 | P a g e
Annex 5: List of PARSE HMM modular domains for M. producens
LIST OF PARSE HMMs HITs for gi|332705439|ref|ZP_08425517.1|/1-2887 amino acid
adenylation domain protein [Moorea producens 3L]
DOMAIN
A
M
T
Cy
A
T
TE
ER
KS
AT
DH
T
Cy
A
T
T
Cy
A
T
Cy
45 | P a g e
AMINO ACID
REGION
997 1253
1427 1938
2079 2146
2171 2610
2786 3000
3189 3253
3275 3528
320 663
823 1261
1383 1697
1768 1943
2079 2146
2171 2610
2786 3000
3189 3253
22 87
150 682
997 1253
2081 2146
2171 2610
SIZE
NUMBER
E-BIT SIZE
228
457
68
450
228
68
267
313
438
327
192
68
450
228
68
68
450
228
68
450
1
1
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
1
5.6e -52
6.1e -124
1.5e -18
7e -159
1.7e -85
3.9e -23
1.2e -43
7.6e -64
4.9e -172
6.1e -121
1.4e -21
2.3e -21
1.8e -172
8.7e -81
8.9e -18
3.6e -10
4.4e -87
7e -51
1.3e -10
3.7e -228