David Hopwood
Lecture 1
(DH1)
Isolation of microbes from soil:
fungi, actinomycetes, other bacteria (left);
streptomycetes (right)
Bioactive compounds from
microbes (2002)
Antibiotics Other
Total
Actinomycetes
7900*
1220
9120
Other bacteria
1400
240
1640
Fungi
2600
1540
4140
Total
11,900
3000
14,900
*70% from Streptomyces spp.
Actinomycetes
Other bacteria
Fungi
Diminishing returns in finding useful natural products
Antibiotic producers are
differentiating microbes
Penicillium
notatum
(penicillin)
Aspergillus
terreus
(lovastatin)
Penicillins
Cephalosporins
Griseofulvin
Valuable
fungal
metabolites
Cyclosporin
Lovastatin
Some
myxobacteria
Myxococcus
Stigmatella
Sorangium
(epothilone)
Myxobacterial metabolites
Epothilone
Et
Me
Me
R
O
OH
R
R
R
E
S
E
S
E
Valuable
myxobacterial
metabolites
Me
S
Me
O
R
OH
S
S
CO 2 H
Ambruticin
A Streptomyces colony on an agar plate
Streptomyces: scanning EM
Young
vegetative
hyphae
Transition
stage:
most
antibiotic
production
Aerial
hyphae,
young spores
Mature
spores
Reproduction
Antibiotic
production
Apoptosis:
nutrient
release
Feeding
Fatal attraction
Needs for new antibiotics
•Overcome acquired resistance:
Staphylococcus aureus (MRSA)
Vancomycin-resistant Enterococcus
MDR and XDR Mycobacterium tuberculosis
Gram-negative respiratory pathogens
•Less toxic anti-viral or anti-cancer agents
•Immunosuppressants, cholesterol lowerers…
How to find new antibiotics
•Novel natural products
•Chemical synthesis
combichem
•Genetics, genetic engineering
Genetics of antibiotic producers
Filamentous fungi:
gene replacements, genomics
Myxobacteria:
transduction, transposon libraries,
gene replacements, genomics…
Streptomyces:
plasmid-mediated conjugation, protoplast fusion,
autonomous and integrating plasmid and phage
cloning vectors, gene replacements,
transposon libraries, genomics…
Streptomyces cloning vectors
Class
Replicon Example
Low copy number autonomous plasmid
SCP2*
pRM5
High copy number autonomous plasmid
pIJ101
pIJ699
Single copy number integrating plasmid
pSAM2
None
pPM927
pSET152
Single copy number integrating phage
ΦC31
KC515
Streptomyces plasmid SCP2
Streptomyces phage C31
Streptomyces mycelium and protoplasts, light microscope
Streptomyces mycelium and protoplasts, electron microscope
The Streptomyces coelicolor
genome
1958
First Streptomyces coelicolor linkage map
First antibiotic gene
(later named act)
1965
antibiotic
bald
white
19901990
1993
(325 clones)
Http://jic-bioinfo.bbsrc.ac.uk/streptomyces - then click “ScoDB II”
LH arm = 1.5 Mb
RH arm = 2.3 Mb
9 May 2002
Core = 4.9 Mb
7825 ORFs
(55 pseudogenes)
63 tRNA genes
6 rRNA operons
72.12% G+C
Isolation of antibiotic
biosynthetic genes
act mutant of Streptomyces coelicolor
(Brian Rudd, 1976)
act mutants of Streptomyces coelicolor
The first act clone
(Francisco Malpartida, 1984)
The act genes of Streptomyces coelicolor
Tailoring steps
Regulation
resistance
Actinorhodin
Chain assembly
(PKS)
Medermycin
(S. AM-7161)
Actinorhodin
(S. coelicolor)
Mederhodin
First
‘hybrid’
antibiotic
(1985)
Manipulation of
polyketide biosynthesis
Some actinomycete antibiotics
(Polyketides)
Medicine
Application Examples
Agriculture
Application Examples
Anti-bacterial Erythromycin Livestock
Tetracyclines rearing
Rifamycin
Anti-cancer
Adriamycin
Monensin
Tylosin
Virginiamycin
Anti-parasitic Avermectin
ImmunoFK 506
suppression
Fungicide
Polyoxin
Kasugamycin
Antifungal
Herbicide
Bialaphos
Candicidin
6-MSA
Cyanidin
Polyketides
Aflatoxin
Oxytetracycline
Erythromycin
Brevetoxin
A fatty acid
Palmitic acid
COOH
Variables in polyketides
(‘Combinatorial biosynthesis’)
Extender
Chirality
Side chains
O
Starter
R
OH
O
OH
OH
Reduction level
KR/DH/ER
H
O
Chain
length
The act and ery PKS gene clusters
KS
Type II PKS
act (simple)
CLF
*
*
Type I
modular PKS
ery
(complex)
DEBS1
LM
Module 1
AT KR
AT ACP KS
Module 2
Module 3
S
DEBS3
Module 5
Module 4
DH ER
AT KR
ACP KS
S
O
DEBS 2
ACP
KS AT ACP KS AT
S
S
AT KR
KR ACP KS
Module 6
TE
AT KR
ACP KS
S
O
O
OH
ACP TE
S
O
S
O
O
O
O
O
HO
HO
O
HO
HO
HO
HO
O
HO
HO
O
HO
HO
O
HO
HO
1
O
OH
OH
6dEB
HO
HO
The DEBS paradigm for complex polyketide biosynthesis
Discovery of ‘cryptic’
secondary metabolites
‘Secondary metabolic’ gene clusters
in Streptomyces coelicolor
3 antibiotics (type II PK, modular PK, NRP)
4 siderophores (2 NRP, 2 other)
3 pigments (type II PK, chalcone, carotenoid)
2 complex lipids (unsaturated FA, hopanoid)
2 signalling molecules (terpenoid, -butyrolactone)
8 other (2 modular PK, 1 NRP, 2 chalcones,
2 terpenoid, 1 deoxysugar)
PK = polyketide, NRP = non-ribosomal peptide, FA = fatty acid
Total length
~ 375 kb
~ 4.5% of the genome
S. coelicolor v. S. avermitilis
Class
S. coelicolor S. avermitilis
Type I PK
3
8
Type II PK
2 (1)
3 (1)
NRP
4
6
Carotenoid
1
1
Desferrioxamine
1
1
Chalcone
3 (1)
1
Others
9
4
Red = similar gene clusters
Enediynes
PKS
Zazopoulos et al. (2003)
Nature Biotech. 21:187
Discoveries/year
Cumulative
discoveries
Watve et al. (2001)
Arch. Microbiol. 176:386
“How many antibiotics are produced by the genus Streptomyces?”
500
current effort level
2003
Total may be 150,000!
“Therefore, by genic manipulation of the cell we have a
means for obtaining, in quantities sufficient for study, many of
the metabolic products of the living organism that would be
otherwise undetectable”
Albert Kelner (1949)
Improvement of productivity
Competing
pathways
Cofactor
availability
Shunt
products
Pathway
genes
Uptake
Export
Product
Substrate
Synthesis
Undesired
substrate
Feedback
inhibition
+ Regulation
Need for functional
genomics
Environmental
factors
Some targets for influencing antibiotic productivity
Many genes
with cumulative effects!
22,000 survivors of mutagenesis
Pick 11 best strains and fuse protoplasts
Pick 7 best from 1000 progeny and fuse protoplasts
Screen 1000 progeny: 2 as good as the best from
1 million cultures screened over 20 year
[Zhang, y. et al. (2002) Nature 415: 644]
Increased productivity of S. fradiae for tylosin
(24,000 colonies screened over 1 year)