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Industrial microbes and products.
George Garrity
September 28, 2006
From A. Kuo and G.M.Garrity 2002 Exploiting Microbial Diversity,
In Biodiversity of Microbial Life, J.T. Staley and A-L Reysenbach, ed,
John Wiley
Examples of microbial products and processes
Hashsham
Marsh
Dale
Asthana
Worden
Alocilja
Saffron
Garrity
2
The search for therapeutic agents
2600 BC Mesopotamia
1500 BC Ebers papyrus
700 drugs
1100 BC China
Wu Shi Er Bing Fang
Prescriptions for 52
diseases
1000 BC Indian Ayurvedic
78 AD Dioscorodies
De Materia Medica
130-200 AD Galen
30 books on pharmacy/medicine
800 AD Avicenna (Ibn Sina)
Persia, first private pharmacy
1745 Curare first collected
1803 Morphine purified
1820 US Pharmacopoeia,
1833 Isolation of codeine
1853 Synthesis of aspirin
1874 Digitalis purified
1901 Epinephrine purified
1903 Barbital synthesized
1920 Ephedrine synthesized
1928 Penicillin described
1938 Crude penicillin purified
1943 Streptomycin described
Bioprospecting
Plants
Animals
Structure
Screening
cultures
FermentationSolvent
extraction
Primary assays
Synthetic compounds
Biocatalysis
Strain
improvement
Chemical
isolation
Dereplication
Biocatalysis
Secondary
assays
Safety
Assessment
Clinical
Trials
Patent
Scale-up
Medicinal
chemistry
Pharmacology
New product
Toxicology
3
Bioprospecting defined
An ancient craft practiced by shamans, priests and
medicine-men
A highly sophisticated, systematic search of nature for
new products
A euphemism for modern screening programs
A politically charged term having different meanings to
developed and developing nations
Evolution of industrial screening
Non-selective isolation, soil main substrate
Antibiotics were major emphasis
Simple, whole organism assays
Major focus on Streptomyces spp, saprophytic fungi
Predominantly secondary metabolites
Streptomyces
octosporusStreptomyces
hygroscopicus
Streptomyces
violaceans
The Golden Age
1945-1960
4
Introduction of mechanism based assays
Expanding the search to pharmacologically active
agents
Expanding the search to “rare genera” of
actinomycetes and other microorganisms
Actinoplanes
regularis
Planomonospora
albus
Actinocorrulia
regularis
Strategic shifts
1960s - 2000
7
Heuristics of natural product screening
Microorganisms produce a vast array of useful products
Chemical diversity is a function of microbial diversity
Diversity of microorganisms is extremely high
The hypothesis
By screening microorganisms broadly many new
products and processes will be discovered.
Ramifications
Reduction to practice
Culture/substrate acquisition:
Microorganisms are a raw materialMicrobiologist collects materials during vacationMicrobiologist’s boss collects material during
vacationBoss’ boss collects material during vacation
Traditional
approach
Culture collections“Purveyors of Fine Cultures”Studies designed to improve recovery of specificgroups
Outside
sources
Use field biologists for collection of specifiedmaterialsAcquire materials collected for other purposes (e.g.petroleum exploration)
Directed
approach
8
Ecologically driven culture isolation
Where do novel microbes the come from?
substrates
the only limitation is your imagination
“Think like a bug!”
How do we get them?
culture isolation strategies
Critical issues
high demand
number of isolates required for screening
when the novelty wears off
monitoring for redundancy
“dereplication”
Casting the net broadly
Types of substrates
Isolation strategies
Nutritional
Physical
Enrichment
Diversity
assessment
What’s
missing?
Variables
Diversity of isolates
Ecological communities
Complexity
Succession
An operational taxonomy to objectively monitor
microbial diversity entering into a screening
program
9
Sediment
(100mg/ml)
SH2O-Y
SH2O-Soil
ChitinGAS
SCHANOB
RAF
R2A
SH2O-Y
SH2O-Soil
Chitin
SC
R2A
SH2O-Y
SH2O-Soil
Chitin
SC
R2A
SH2O-Y
SH2O-Soil
Chitin
SC
R2A
Primary
isolationSecondary
isolation
Gent
5mg/ml
Sediment
(100mg/ml)
Rif
5mg/ml
Novo
25mg/ml
Incubation
50d @ 27C
benomyl
7ppm
30% 70%
A typical isolation strategy
Simplified dereplication
Variation of table-top sorting
Isolation plates scanned at 400X and 1000X
ELWD objectives/transmitted illumination
Selection criteria
Transferred to YME plate
Culture purity
8 isolates/100mm plate
Transferred to EYES
Single isolate/60 mm
Limitations
Non-cumulative data
Comparisons within/between samples
10
A sampling of Actinobacteria from marine sediments
Technique
Restriction fragment length polymorphism (RFLP)Low frequency restriction fragment analysis (PFGE)
RibotypingDNA amplification (AFLP, AP-PCR, rep-PCR, RAPD, ...)
Phage and bacteriocin typingSerological (monoclonal, polyclonal) techniques
Zymograms (multilocus enzyme polymorphism)Total cellular protein electrophoretic patternsDNA-DNA hybridizations% G+C
tDNA-PCRChemotaxonomic markers (polyamines, quinones, ...)Cellular fatty acid fingerprinting (FAME)Cell wall structurePhenotype (classical, API, Biolog, ...)rRNA sequencingDNA probes (ISH & FISH)DNA sequencing
Stra
inSpe
cies
Gen
us
Family
DNA amplification (ARDRA)
Adapted from Gillis et al., Polyphasic Taxonomy., Bergey’s Manual of Systematic Bacteriology, Second Edition., 2001
11
Application of ecological concepts
Diversity
a key concept
patterns of spatial/temporal distribution
indication of ecosystem “wellness”
Two components
variety (species richness)
abundance
Diversity measures
need to look at both components
Communities
distribution of interacting species in a defined ecosystem
Species distributions
1
10
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Geometric
Log
Log Normal
Species sequence
Ab
un
da
nce
(%
)
12
1 3 5 7 9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
Soil-30
Soil-5
Soil-12
Soil-25
Soil-29
Soil-41
Soil-45
Soil-44
0
50
100
Species rank 43
Abundance
The rank abundance plot
13
Does the strategy still work?
From: Lam, KS 2006 Curr Opin. Microbiol 8:252
NCEs from marine Actinobacteria
From: Lam, KS 2006
Curr Opin. Microbiol 8:252
15
The end of the second wave. Will there be a third?
Shift of discovery from big pharma to small
biotech organizations
Application of state-of-the-art technology
Combinatorial chemistry
Discovery/development platforms
Genomic approaches
The realities of science as a business
High capital outlay
Treatment of acute vs chronic disease
The great
experiment
16
Current state of affairs in natural product screening
From: Barrett, JF 2005 Curr Opin. Microbiol 8:498
Projected new chemical and molecular entities
From: Barrett, JF 2005 Curr Opin. Microbiol 8:498
17
The end of the second wave. Will there be a third?
Shift of discovery from big pharma to small
biotech organizations
Application of state-of-the-art technology
Combinatorial chemistry
Discovery/development platforms
Genomic approaches
The realities of science as a business
High capital outlay
Treatment of acute vs. chronic disease
Exploiting the yet-to-be cultivated
Discovering novel products/pathways
Physiological insights
Ecological insights
The great
experiment
Metagenomics as
a new discovery
tool
18
Microbial genomics and natural product biosyntehsis..
From: Van Lanen and Shen 2006 Curr Opin. Microbiol 9:252
Some examples of NCE discovered based on genomic techniques
From: Van Lanen and Shen 2006 Curr Opin. Microbiol 9:252
19
What have we learned in 60+ years?
Natural products continue to hold promise
Rate of discovery continues to be high
Genomic/metagenomic approaches promise
to significantly extend the number of NCE
However,
Novel chemistry doesn’t always yield novel
drugs
Safety and efficacy only partially
correlated with structure
Costs associated with development are
a significant barrier to success
We are completing a circle
Renewed interest/emphasis on
cultivation and pure culture methods
Natural product
discovery
It’s time to put on your reviewer’s hat…
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