Industrial microbes and products.
George Garrity
September 28, 2006
Examples of microbial products and processes
Asthana
Dale
Hashsham
Marsh
Saffron
Garrity
Worden
Alocilja
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
1
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)
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
Persia, first private pharmacy
Screening
cultures
Patent
Bioprospecting
Scale-up
New product
Plants
Animals
Fermentation
Solvent
extraction
Strain
improvement
Clinical
Trials
Chemical
isolation
Safety
Assessment
Toxicology
Primary assays
Structure
Medicinal
chemistry
Biocatalysis
Secondary
assays
Synthetic compounds
Pharmacology
Biocatalysis
Dereplication
2
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
The Golden Age
1945-1960
Streptomyces
violaceans
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
hygroscopicus
Streptomyces
octosporus
3
Strategic shifts
1960s - 2000
Actinoplanes
regularis
Introduction of mechanism based assays
Expanding the search to pharmacologically active
agents
Expanding the search to “rare genera” of
actinomycetes and other microorganisms
Planomonospora
albus
Actinocorrulia
regularis
4
5
6
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:
Traditional
approach
Microorganisms are a raw material
Microbiologist collects materials during vacation
Microbiologist’s boss collects material during
vacation
Boss’ boss collects material during vacation
Outside
sources
Culture collections
“Purveyors of Fine Cultures”
Studies designed to improve recovery of specific
groups
Directed
approach
Use field biologists for collection of specified
materials
Acquire materials collected for other purposes (e.g.
petroleum exploration)
7
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
Variables
Types of substrates
Isolation strategies
Nutritional
Physical
Enrichment
Diversity
assessment
Diversity of isolates
Ecological communities
Complexity
Succession
What’s
missing?
An operational taxonomy to objectively monitor
microbial diversity entering into a screening
program
8
A typical isolation strategy
Primary
isolation
Secondary
isolation
Sediment
(100mg/ml)
Sediment
(100mg/ml)
SH2O-Y
SH2O-Soil
Chitin
GAS
SC
HANOB
RAF
SH2O-Y
SH2O-Y
SH2O-Y
SH2O-Soil
Chitin
SC
SH2O-Soil
Chitin
SC
R2A
R2A
SH2O-Soil
Chitin
SC
R2A
R2A
benomyl
7ppm
30%
Gent
5mg/ml
Rif
5mg/ml
Incubation
50d @ 27C
Novo
25mg/ml
70%
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
9
Technique
Fa
m
i
G ly
en
u
Sp s
ec
St ies
ra
in
A sampling of Actinobacteria from marine sediments
Restriction fragment length polymorphism (RFLP)
Low frequency restriction fragment analysis (PFGE)
Phage and bacteriocin typing
Serological (monoclonal, polyclonal) techniques
Ribotyping
DNA amplification (AFLP, AP-PCR, rep-PCR, RAPD, ...)
Zymograms (multilocus enzyme polymorphism)
Total cellular protein electrophoretic patterns
DNA-DNA hybridizations
% G+C
DNA amplification (ARDRA)
tDNA-PCR
Chemotaxonomic markers (polyamines, quinones, ...)
Cellular fatty acid fingerprinting (FAME)
Cell wall structure
Phenotype (classical, API, Biolog, ...)
rRNA sequencing
DNA probes (ISH & FISH)
DNA sequencing
Adapted from Gillis et al., Polyphasic Taxonomy., Bergey’s Manual of Systematic Bacteriology, Second Edition., 2001
10
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
Abundance (%)
100
Geometric
Log
10
Log Normal
1
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
Species sequence
11
100
50
Soil-44
Soil-45
0
Soil-41
rank
Soil-5
Soil-30
43
cies
Soil-25
Soil-12
39
Spe
Soil-29
41
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
Abundance
The rank abundance plot
12
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
13
Bioactive volatile organic compounds
From: Strobel, G. 2006 Curr Opin. Microbiol 9:240
14
The end of the second wave. Will there be a third?
The great
experiment
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
15
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
16
The end of the second wave. Will there be a third?
The great
experiment
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
Metagenomics as Exploiting the yet-to-be cultivated
a new discovery
Discovering novel products/pathways
tool
Physiological insights
Ecological insights
17
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
18
What have we learned in 60+ years?
Natural product
discovery
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
It’s time to put on your reviewer’s hat…
19