david hopwood lecture 1 (dh1). isolation of microbes from soil: fungi, actinomycetes, other bacteria...
TRANSCRIPT
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.
Diminishing returns in finding useful natural products
ActinomycetesOther bacteriaFungi
Antibiotic producers aredifferentiating microbes
Penicilliumnotatum
(penicillin)
Aspergillusterreus
(lovastatin)
Penicillins Cephalosporins
Griseofulvin
Cyclosporin Lovastatin
Valuablefungal
metabolites
Myxococcus
Sorangium(epothilone)
Stigmatella
Somemyxobacteria
Myxobacterial metabolites
Epothilone
Ambruticin
Valuablemyxobacterial
metabolites
Et
Me
MeMe
Me
CO 2H
O
OH
OH
O
EE
ES
S
S
R
R
S
SR
R
R
A Streptomyces colony on an agar plate
Streptomyces: scanning EM
Young
vegetative
hyphae
Transition
stage:
most
antibiotic
production
Aerial
hyphae,
young spores
Mature
spores
Apoptosis:nutrientrelease
Feeding
Antibioticproduction
Reproduction
Fatal attraction
Needs for new antibioticsNeeds for new antibiotics•Overcome acquired resistance:
Staphylococcus aureus (MRSA)Vancomycin-resistant EnterococcusMDR and XDR Mycobacterium tuberculosisGram-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
Filamentous fungi:gene replacements, genomics
Myxobacteria:transduction, transposon libraries,gene replacements, genomics…
Streptomyces:plasmid-mediated conjugation, protoplast fusion,autonomous and integrating plasmid and phagecloning vectors, gene replacements, transposon libraries, genomics…
Genetics of antibiotic producers
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 cloning vectors
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
1965
First antibiotic gene(later named act)
1990 1990
antibioticbaldwhite
1993
Http://jic-bioinfo.bbsrc.ac.uk/streptomyces - then click “ScoDB II”
(325 clones)
LH arm = 1.5 Mb
RH arm = 2.3 Mb
Core = 4.9 Mb
7825 ORFs(55 pseudogenes)
63 tRNA genes6 rRNA operons
72.12% G+C
9 May 2002
Isolation of antibioticbiosynthetic 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
Actinorhodin
Tailoring steps Regulationresistance
Chain assembly(PKS)
Actinorhodin(S. coelicolor)
Medermycin(S. AM-7161)
Mederhodin
First‘hybrid’
antibiotic(1985)
Manipulation of polyketide biosynthesis
Some actinomycete antibiotics(Polyketides)
Medicine Agriculture
Application Examples Application Examples
Anti-bacterial ErythromycinTetracyclinesRifamycin
Livestockrearing
MonensinTylosinVirginiamycin
Anti-cancer Adriamycin Anti-parasitic Avermectin
Immuno-suppression
FK 506 Fungicide PolyoxinKasugamycin
Antifungal Candicidin Herbicide Bialaphos
Polyketides
Palmitic acid
6-MSACyanidin
Erythromycin
Oxytetracycline
Brevetoxin
Aflatoxin
COOHA fatty acid
Variables in polyketides
OH
O
RStarterO
Side chains
Extender
Chainlength
(‘Combinatorial biosynthesis’)
OH
Reduction level
KR/DH/ER
OH OH
Chirality
Type II PKSact (simple)
Type Imodular PKS
ery (complex)
The act and ery PKS gene clusters
*
*
CLFKS
AT ACP
AT
ACPKS
KR AT
ACPKS
KR
AT ACPKS AT ACPKS KR
DH ER AT
ACPKS
KR AT
ACPKS
KR
TE
O
O
O
OH
OH
OH
Module 1Module 2
Module 3Module 6Module 4
Module 5LM
DEBS1 DEBS 2 DEBS3
6dEB
SO
SO
HO
HO
SO
HO
HO
HO
SO
O
HO
HO
O
SO
HO
HO
O
SO
HO
HO
HO
O
HO
SO
HO
1
TE
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
Cumulativediscoveries
Watve et al. (2001) Arch. Microbiol. 176:386 “How many antibiotics are produced by the genus Streptomyces?”
500
current effort level
Increased effort
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
Regulation
+ -
Environmental factors
Cofactoravailability
Shunt products
Feedbackinhibition
Pathway genes
Substrate Product
Uptake Export
Competing pathways
Synthesis
Undesiredsubstrate
Some targets for influencing antibiotic productivity
Need for functionalgenomics
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 from1 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)