Screening for Potential Microorganisms

General consideration of screening

• Screening can never be considered a routine activity.

• In order to be successful, screening must be an interdisciplinary activity.

• There are no universal screening methods.

Screening can never be considered a routine activity

• The methods must always be adapted to the newest techniques and knowledge.

• The goal is always to detect and identify new substances of commercial interest and to separate them in the quickest possible way from the numerous easily detected substances that are of no commercial interest.

In order to be successful, screening must be an interdisciplinary activity• (1) Microbiology is involved in the isolation and

identification of microorganisms, strain preservation, testing for biological activity, and fermentation practice.

• (2) Biochemistry provides the analytical procedures needed as well as the approaches to purification of the biologically interesting molecules.

• (3) Synthesis of substrates and inhibitors falls under the purview of the chemist.

• (4) The bibliographic specialist searches the literature as well as the various computerized data bases.

• (5) The engineer's activity focuses on the development of technical equipment needed for the successful process.

There are no universal screening methods.

• The success of a screening program depends on both the kinds of organisms used and the methods for detection of activity.

• Currently, the choice of strain has a 30 - 40% influence on the outcome, the test procedure a 60-70% influence.

Current focuses of most screening programs

• Chemotherapeutically useful products for the following areas: activity against antibiotic-resistant strains, tumors, fungi and viruses

• Enzyme inhibitors and pharmacologically active substances (hormones, etc.)

• Better starter cultures for the food industry • Microorganisms that are capable of degrading

hazardous and persistent chemicals

Strains used in screening • Microorganisms isolated from nature, sometimes

from a special, ecological niche that may not even exist in a particular country

• Microorganisms obtained from a culture collection• Microorganisms used for preparing native

fermented food products

Strategies for the isolation of a suitable industrial microorganism from the environment • Shotgun approach. Samples of free living microo

rganisms, biofilms or other microbial communities are collected from animal and plant material, soil, sewage, water and waste streams, and particularly from unusual man-made and natural habitats. These isolates are then screened for desirable traits.

• Objective approach. Take samples from specific sites where organisms with the desired characteristics are considered to be likely components of the natural microflora.

Isolation of strains from natural sources

1. Numerous strains to be exploited from various habitats

• A gram of soil contains between 106-108 bacteria, 104-106 actinomycete spores, and 102-104 fungal spores.

• Less than 1% of the world's microorganisms have been intensively studied.

• Above all, the approximately 100,000 known fungi have been poorly studied, so that a vast number of new natural products can be expected from this group in the future.

Isolation of strains from natural sources

2. Isolate strains from extreme or unusual environments

• Hope such strains may be capable of producing new metabolites.

• For instance, microorganisms from high altitudes, cold habitats, sea water, deep sea, deserts, geysers, and petroleum fields are being examined

Isolation of strains from natural sources

3. Enrichment of special types of microorganisms / appropriate selective media and growth conditions

Isolation of strains from natural sources

4. General scheme of isolation of strains

• Samples suspended in sterile water containing Tween. The sample is vigorously agitated

• The supernatant is diluted 10-1-10-10 • Diluted samples are plated on various culture me

dia and then incubated. (choosing or developing the appropriate selective media and growth conditions)

• Picked single colonies are “re-streaking” • Maintained pure strains are as agar slant cultures

Isolation of strains from natural sources

5. Testing the initial isolates directly • The screening procedure can often be speeded

up by testing the initial isolates directly for biological activity. The soil or water samples are diluted directly onto the test plates and only those colonies showing activity are isolated.

• Some examples of procedures that can be carried out directly on agar plates are given in Table 2.2.

Isolation of strains from natural sources

6. Continuous cultivation • Provide a method to isolate microorganisms fro

m mixed cultures with improved properties. e.g. thermophilic or alcohol tolerant strains

Isolation of strains from natural sources

7. Variability of metabolites and variability of genera

• The variability of metabolites produced by individual genera is somewhat limited except in the streptomycetes.

• Except for industrial enzymes, Bacillus strains almost exclusively produce peptide antibiotics.

• In one extensive study, 20,000 Actinoplanes strains were isolated, of which 13,000 were screened for the formation of antibiotics. Within a ten year span, 41 new antibiotics were isolated and characterized from these strains. These antibiotics turned out to be almost all either acetylmalonyl or amino acid derivatives.

Isolation of strains from natural sources

8. Goal of first level screening• Activity or concentration of the target product

per se is not of major concern.• Other important features, such as stability and,

where necessary, non-toxicity must also be screened.

Isolation of strains from natural sources

9. Pure culture or consortia?

Test systems for the screening programs

• Intelligent screening methods: Omura’s works, squalene synthase inhibitor etc.

• Unusual microorganisms: discovery of avermectins

• Instrumental aids: FPLC

42 completely new compounds were found by Omura (1986) • Using systems that detected microbially produced

substances with antibacterial, antimycoplasmal, antianaerobe, antifungal, antiparasite, and antitumor activity.

• Substances were also found that acted as herbicides and as inhibitors of penicillin, elastase, and adenosine deaminase.

Omura et al. 1979. discovered inhibitors of cell wall biosynthesis 1. Inhibited Bacillus subtilis but did not inhibit Ach

oleplasma laidlawii (lacks a cell wall)2. Inhibited the incorporation of meso-diaminopime

lic acid but did not inhibit the incorporation of leucine

3. Eliminated substances whose molecular weights were greater than 1,000.

4. Azureomycin was discovered from culture filtrates of 10,000 strains (fungi, bacteria and actinomycetes).

The discovery of avermectins in the early 1980s: • The selection of microorganisms was biased toward

those with unusual morphological traits and nutritional requirements

• Administered fermentation broths in the diet to mice infested with the nematode Nematospiroides dubius. (expensive assay, but simultaneously tested for efficacy against the nematode and toxicity to the host)

• Streptomyces avermitilis (morphological characteristics were unlike those of other known Streptomyces species) produces a family of closely related macrocyclic lactones that are active against certain nematodes and arthropods at extremely low doses but have relatively low toxicity to mammals

• These avermectins are highly effective in veterinary use and show promise for treating infestations in humans

Avermectin B1. The major

macrocyclic lactone produced

by S. avermitilis.

Selective inhibition of cholesterol biosynthesis - 1 • Isopentenyl-, geranyl-, and farnesylpyrophosphate

are precursors of sterols and other isoprenoid derivatives

• Mevinolin (from Aspergillus terreus) and compactin (from Penicillium spp.) are highly effective to reduce serum cholesterol in humans. They are potent inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase and block formation of all the products of polyisoprenoid pathway

Selective inhibition of cholesterol biosynthesis - 2 • Squalene synthase is an attractive target for selecti

ve inhibition of cholesterol biosynthesis. Zaragozic acid A was obtained from an unidentified fungus found in a water sample taken from the Jalon river in Zaragoza, Spain ─ hence the name. Soon after, zaragozic acid B and C were obtained from fungi isolated elsewhere: Sporomiella intermedia and Leptodontium elatius, respectively

Finding NAD(H)-dependent dehydrogenases for the reduction of prochiral 2-ketoacids to the corresponding chiral 2-hydroxyacids • Lactic acid bacteria• Cultivated anaerobically• Centrifugation, disintegration, high-speed centrifug

ation, filtration ((0.2m)• Crude extract to fast protein liquid chromatography

(FPLC) (Instrumental aids)• Enzyme assay: 2-ketoacids, NADH, buffer, decreas

e in A340

References

• David Perlman, 1980• Parton and Willis, 1990• Waites, M. J., N. L. Morgan, J. S. Rockey, and G.

Higton. 2001. Industrial Microbiology: An Introduction. Blackwell Science Ltd

問題

• 如何才能快速有效篩選獲得有用的微生物？ • 下列名詞的內涵？ FPLC, agents against antibio

tic-resistant strains, starter