1

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

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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

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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

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6

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

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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

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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

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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

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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

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Bioactive volatile organic compounds

From: Strobel, G. 2006 Curr Opin. Microbiol 9:240

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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

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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

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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

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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

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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…