The following points highlight the five main steps involved in developing producer strains. The steps are: 1. Isolation of Industrial Microorganisms 2. Screening for New Products 3. Identification of Metabolites 4. Maintenance of Microbial Isolates 5. Strain Improvement.
Step # 1. Isolation of Industrial Microorganisms:
The first step in developing producer strains is the isolation of concerned microorganisms from their natural habitats. Alternatively, microorganisms can be obtained as pure cultures from organisations, which maintain culture collections, e.g.. American Type Culture Collection (ATCC), Rockville, Maryland, U.S.A.; Commonwealth Mycological Institute (CMI), Kew, Surrey, England; Fermentation Research Institute (FERM), Tokyo, Japan; Research Institute for Antibiotics (RIA), Moscow, Russia etc.
The microorganisms of industrial importance are, generally, bacteria, actinomycetes, fungi and algae. These organisms occur virtually everywhere, e.g., in air, water and soil, on the surfaces of plants and animals, and in plant and animal tissues. But most common sources of industrial microorganisms are soils, and lake and river mud.
Often the ecological habitat from which a desired microorganisms is more likely to be isolated will depend on the characteristics of the product desired from it, and of process development. For example, it the objective is to isolate a source of enzymes, which can withstand high temperatures, the obvious place to look will be hot water springs.
A variety of complex isolation procedures have been developed, but no single method can reveal all the microorganisms present in a sample.
Many different microorganisms can be isolated by using specialized enrichment culture techniques, e.g., soil treatment (UV irradiation, air drying or heating at 70-120°C, filtration or continuous percolation, washings from root system, treatment with detergents or alcohols, pre-incubation with toxic agents), selective inhibitors (antimetabolites, antibiotics, etc.), nutritional variations (specific C and N sources), variations in pH, temperature, aeration, etc.
The enrichment culture techniques are designed for selective multiplication of only some of the microorganisms present in a sample. These approaches, however, take a long time (20-40 days), and require considerable labour and money.
Obviously, the procedures for isolation of actinomycetes, algae, bacteria and fungi differ markedly, and they usually utilize specialized media.
The main isolation methods used routinely for isolation from soil samples are as follows: sponging (soil directly), dilution gradient plate, aerosol dilution, flotation, and differential centrifugation. Often these methods are used in conjunction with an enrichment culture technique.
Step # 2. Screening for New Products:
The next step in developing producer strains after isolation of microorganisms is their screening. A set of highly selective procedures, which allows the detection and isolation of microorganisms producing the desired metabolite constitutes primary screening. Ideally, primary screening should be rapid, inexpensive, predictive, specific but effective for a broad range of compounds and applicable on a large scale.
Primary screening is time-consuming and labour intensive since a large number of isolates have to be screened to identify a few potential ones. However, this is possibly the most critical step since it eliminates the large bulk of unwanted useless isolates, which are either non-producers or producers of known compounds.
The need for the latter would become obvious in the light of the fact that till 1987 more than 3,000 different metabolites were well characterised, and every year about 100 new ones are added to this list.
Therefore, rapid and accurate determination of new metabolites is necessary to avoid a wasteful duplication of effort. Computer-based databases play an important role by instantaneously providing detailed information about the already known microbial antibiotic compounds.
Rapid and effective screening techniques have been devised for a variety of microbial products, which utilize either a property of the product or that of its biosynthetic pathway for detection of desirable isolates.
Some of the screening techniques are relatively simple, e.g., for extracellular enzymes and enzyme inhibitors. However for most microbial products of high value, the screening is usually complex and tedious, and often may involve two or more steps, e.g., for antimicrobials. Table 39.1 presents a brief and generalized summary of the popular screening approaches of some classes of compounds, and a detailed consideration is not attempted.
In some cases, it may be desirable to concentrate on a group of organisms expected to yield new products. For example, the search for new antibiotics now focusses on rare actinomycetes, i.e., Actinomycetes other than those belonging to the genus Streptomyces, which make up <5% of total actinomycetes population in soil.
Suitably designed specialized screening techniques may be used to detect compounds having various pharmacological activities (other than antibiotics), some of which are listed in Table 39.2. The discovery of new microbial products may, in fact, be thought to be limited by the availability of specific, precise and rapid screening techniques for them.
Step # 3. Identification of Metabolites:
The initial screening is ordinarily done in plates using agar media. Subsequently, desirable isolates are screened in liquid cultures using shaker vessels since the results from plate arrays can not always be reproduced in shake cultures. Shake cultures also allow the evaluation of different fermentation conditions, and are amenable to automated detection systems.
This step allows the identification of desirable isolates and the suitable culture conditions for them. In the next step, the cultures are scaled up to 10-70 litres; these cultures provide the material necessary for developing a suitable procedure for isolation of the active principle or compound of interest by trying out various solvents-extraction procedures over a wide range of pH.
Several other types of data, e.g., adsorption of the metabolite on ion exchange resins, its stability to pH and heat, etc., are also collected. During chemical purification, the biological activities of various preparations are assayed using a cup-plate or paper disc assay.
The bioactivity of a metabolite is initially identified using paper chromatography or thin-layer chromatography; more recently, high performance liquid chromatography (HPLC) is more commonly and profitably used. Once the metabolite is reasonly purified, its chemical nature is determined. This information allows the researcher to decide if this metabolite in a new one.
Step # 4. Maintenance of Microbial Isolates:
Industrial microbiology continuously uses specific microorganism isolates/strains as research, assay, development and production cultures. These strains are highly valuable and must be preserved over long periods without any genetic and, as a result, phenotypic changes.
A microbial strain discovered to produce a useful product is called research culture since it is now used for studies on product isolation and identification, strain improvement, etc. The microorganisms used for assays of the biological activities of microbial products as well as for various screening techniques are known as assay cultures.
A research culture producing a metabolite of sufficient commercial interest is used for developing the large scale fermentation procedures; it is now termed as development culture. These cultures become production cultures if their product is good enough to be marketable.
The various cultures can be maintained for reasonably prolonged periods using one of the following approaches:
1. Low temperature storage at 2-6°C on agar slants or in liquid medium; it is useful for relatively shorter periods (2-6 months) for working stock cultures.
2. Storage as frozen cultures at -20 to -100°C (both agar slants and liquid cultures).
3. Storage as lyophilized cells under vacuum at low temperatures (5°C or even -20 to -70°C). Lyophilization removes the free water from cells and spores. Protective agents like skimmed milk or sucrose reduce the detrimental effects of lyophilization. Lyophilization is easily done by using automated freeze-drying machines in which the cells are first frozen and then their free water is withdrawn under high vacuum.
4. Storage of vegetative cells/spores in liquid nitrogen (-196°C) or in the vapour phase of liquid nitrogen (-167°C).
Preservation of lyophilized cells is the most widely used method, but storage in liquid nitrogen is also as effective and widely used. These methods can, theoretically, maintain the microorganisms indefinitely.
5. Air dried at room temperature on sterile loam sand or on other natural substrates like maize seed, rice bran, etc., and stored at room temperature or in a refrigerator.
Bacterial cultures may remain viable up to 70-80 years. It is more suited for organisms that form spores.
6. Storage in glycerine stabs (0.85 ml of cell suspension mixed with 0.15 ml of sterile glycerol and stored at -70 or -75°C). This is commonly used for recombinant bacteria.
Procedure listed in item 1 for culture maintenance is used for working stock cultures, while those listed at items 2 to 6 are used for primary stock cultures. Working stock cultures are maintained in a vigorous and uncontaminated condition and are used quite frequently. They must be checked frequently for characteristic features and contamination.
In contrast, primary stock cultures are kept in long-term storage for later use; they are used to produce new working stock cultures, as per need. A stock culture may be simply defined as a culture, which serves as a source of inoculum.
Step # 5. Strain Improvement:
After an organism producing a valuable product is identified, it becomes necessary to increase the product yield from fermentation to minimise production costs.
Product yields can be increased by the following strategies:
(i) Developing a suitable medium for fermentation,
(ii) Refining the fermentation process, and
(iii) Improving the productivity of the strain.
Generally, major improvements arise from the last approach; therefore, all fermentation enterprises place a considerable emphasis on this activity. The techniques and approaches used to genetically modify strains to increase the production of the desired product is called strain improvement or strain development.
Strain improvement is based on the following three-approaches:
(i) Mutant selection and selective isolation of mutants (secondary screening),
(ii) Recombination, and
(iii) Recombinant DNA technology (Table 39.3).
1. Mutant Selection and Selective Isolation of Mutants (Secondary Screening):
Large scale mutant selection programmes begin when favourable reports of clinical trials are obtained. In the early stages, selection of spontaneous mutants may be helpful, but induced mutations are the most common sources of improvements.
Mutations occurring without any specific treatment are called spontaneous mutation, while those resulting due to a treatment with certain agents are known as induced mutations, the agents being referred to as mutagens.
Application of mutagens to induce mutations is called mutagenesis. Either physical or chemical mutagens can be employed. Usually, the frequency of mutants with desirable phenotype is quite low; hence the major bottleneck is the identification and isolation of such cells from among the large number of non- mutant/undesirable mutant cells.
A mutation is a sudden and heritable change in the traits of an organism. Many mutations bring about marked changes in the biochemical characters of practical interest; these are called major mutations. Some of the major mutations can be useful in strain improvement.
For example, the original strain of Streptomyces griseus produced small amounts of streptomycin and large amounts of mannosidostreptomycin, which has low antibiotic activity.
A major mutant isolated from this strain produced negligible amounts of mannosidostreptomycin and much larger quantities of streptomycin. Similarly, a mutant strain (S-604) of Streptomyces aureofaciens produces 6-demethyl tetracycline in place of tetracycline; this demethylated form of tetracycline is the major commercial form of tetracycline.
In contrast, most improvements in biochemical production have been due to the stepwise accumulation of so called minor genes. These genes lead to small increases (or decreases) in the antibiotic or other biochemical production, and selection may be expected to result in a 10-15% increase in yield.
The selected strains are usually subjected to successive cycles of mutagenesis and selection, and after several cycles large increases in yields are likely to be obtained.
In some cases, improvements have been obtained even without the use of mutagens. Mutants of Penicillium chrysogenum were selected for increased penicillin production; each cycle of selection was preceded by mutagen (chemical) treatment and resulted in only small changes in penicillin yield. But after several (about a dozen) cycles of selection, a strain (E15-1) was obtained that yielded 55% more penicillin than the original strain (Fleming Strain).
A majority of desirable mutants, especially the ‘minor gene’ mutants, showing increased production, are isolated by screening a large number of clones surviving the mutagen treatment; this is called selective isolation of mutants (secondary screening). But this approach requires a large amount of work.
Therefore, efforts have increasingly focussed on developing techniques for the isolation of particular classes of mutants, which are likely to be overproducers (Table 39.3).
Some of the relevant strategies are briefly summarised below; the selection for these classes of mutants is simple, easy and effective:
(i) Isolation of auxotrophic mutants is the basis for commercial amino acid production in Japan from the bacterium Corynebacterium glutamicus. An auxotrophic mutant has a defect in one of its biosynthetic pathways so that it requires a specific biochemical for normal growth and development.
For example, phe– mutants require phenylalanine for growth; such mutants of C. glutamicus accumulate tyrosine. Similarly, tyr– mutants accumulate phenylalanine, while phe– + tyr– mutants accumulate tryptophan.
(ii) Many analogue-resistant mutants have feed-back insensitive enzymes of the biosynthetic pathway the analogue of whose product was used for selection of such cells. In feed-back inhibition, activity of an enzyme is inhibited by the end-product of the biosynthetic pathway in which the enzyme participates.
For example, when tyr– mutants of C. glutamicus were selected for resistance to 50 mg/l p- fluorophenylalanine (analogue of phenylalanine), there was a nearly 7-fold increase in phenylalanine accumulation over that of the tyr– mutant.
(iii) Sometimes revert-ants from nonproducing mutants of a strain are high producers, e.g., one such reversion mutant of Streptomyces viridifaciens showed over 6-fold increase in chlortetracycline production over the original strain from which the nonproducing mutant was obtained. When a mutant mutates back to its original phenotype it is called reversion, and the mutant is known as revertant, e.g., non-producer mutant mutating back to producer phenotype.
(iv) Reversion mutants of appropriate auxotrophs may often be high producers, e.g., in case of S. viridifaciens reversion mutants of an auxotrophic mutant requiring homocysteine showed 28% more chlortetracycline yield than the original strain.
(v) In some cases, selection for resistance to the antibiotic produced by the organism itself may lead to increased yields. For example, Streptomyces aureofaciens mutants selected for resistance to 200-400 mg/l chlortetracycline showed a four-fold increase in the production of this antibiotic.
(vi) Sometimes, mutants with altered cell membrane permeability show high production of some metabolites. A mutant E. coli strain has defective lysine transport; it actively excretes L-lysine into the medium to 5-times as high concentration as that within its cells.
(vii) Mutants have been selected to produce altered metabolites, especially in case of aminogycoside antibiotics. For example. Pseudomonas aureofaciens produces the antibiotic pyrrolnitrin; a mutant of this organism yields 4′-fluoropyrrolnitrin.
The above and many other approaches for selection of mutants can be most profitably used when the biosynthetic pathway for the concerned product is known, as are the precursors and the regulatory mechanisms.
Mutant selection has been the most successful approach for strain improvement, but major advances are being made in the exploitation of other strategies, i.e., recombination and recombinant DNA technology.
2. Recombination:
Recombination may be defined as formation of new gene combinations among those present in different strains. This approach has been highly successful in the improvement of animals and plants.
Once several different mutants have been isolated, recombination is used for both genetic analysis as well as strain improvement. It is used to bring the desirable alleles present in two or more strains into a single strain to increase product yields or to generate new products.
Recombination may be based on:
(i) Sexual reproduction,
(ii) Parasexual cycle or
(iii) Protoplast fusion (Table 39.3).
(i) Sexual Reproduction:
Conjugation, mediated by sex-factor, occurs in many bacteria and actinomycetes, including Streptomyces. Conjugation leads to the formation of usually partial diploids in which crossing over produces recombinant genotypes. Recombination are recovered and used for genetic studies like linkage mapping.
Similarly, yeast has two mating types; the cells of opposite mating types fuse to form diploid heterozygous cells (which are non-mating). The diploid cells undergo meiosis to produce four haploid spores, which give rise 10 vegetative cells.
(ii) Parasexual cycle:
Most industrially important fungi are asexual. However, their haploid hyphae often fuse to produce heterokaryons, i.e., cells having two distinct nuclei. Sometimes, the two nuclei of heterokaryons fuse and produce diploid nuclei.
Occasionally, mitotic recombination coupled with mitotic reduction yields haploid nuclei from the diploid ones giving rise to recombinants. In some cases, attempts have been made to use parasexuality for strain improvement, e.g., in Penicillium chrysogenum.
(iii) Protoplast fusion:
Protoplasts of bacteria, actinomycetes and fungi are isolated by treatment with a variety of lytic enzymes. An osmoticum is necessary for protoplast stability, and fusion is usually induced by PEG (polyethylene glycol) treatment. Protoplast fusion has been used to produce Cephalosporium acremonium strains, which yield significantly higher cephalosporin C; this was done by fusing protoplasts of auxotrophic mutants.
It may be noted that this organism had not responded well to mutant selection programmes. Similarly, P. chrysogenum strains producing low p-hydroxypenicillin were developed by protoplast fusion; this is important because it interferes with the formation of cephalosporin.
Protoplast fusion between nonproducing strains of two species, Streptomyces griseus and Streptomyces tenjimariensis, has yielded a strain that produces indolizomycin, a new indolizine antibiotic. Usually recombinants are recovered from protoplast fusion products, and some of them may possess desirable features.
3. Recombinant DNA Technology:
Recombinant DNA Technology involves the isolation and cloning of genes of interest, production of the necessary gene constructs using appropriate enzymes and then transfer and expression of these genes into an appropriate host organism.
This approach is also called genetic engineering.
This technique has been used to achieve the following two broad objectives:
(i) production of recombinant proteins, and
(ii) modification of the organism’s metabolic pattern for the production of new, modified or more quantity of metabolites (metabolic engineering).
(i) Recombinant Proteins:
Recombinant proteins are the proteins produced by the transferred gene or transgene, and they themselves are of commercial value, e.g., insulin, interferon, etc., produced in bacteria, etc.
(ii) Metabolic Engineering:
When metabolic activities of an organism are modified by introducing into it transgenes which affect enzymatic, transport and/or regulatory functions of its cells, it is known as metabolic engineering.
The approaches for metabolic engineering are numerous, some of which are briefly summarised below (Table 39.4) to give an outline of these approaches and their potential consequences. It is imperative for metabolic engineering that the biosynthetic pathways to be modified and their regulatory controls are well known, and the genes involved are identified and cloned.
(a) A transgene may be added, which encodes an enzyme to modify a metabolite produced by the organism to yield a new product of interest. For example, Acremonium chrysogenum produces cephalosporin C. The gene encoding D-amino acid oxidase from Fusarium was introduced into A. chrysogenum. This enzyme converts cephalosporin C into 7-amino-cephalosporanic acid, a precursor of several semisynthetic antibiotics.
(b) The enzyme encoded by transgene may enable a better utilization of the substrate or even of previously inaccessible components of the substrate. For example, normal yeasts are unable to utilize cyclodextrins present in the malt; this increases the calorie content of beer. Transgenic yeasts capable of utilizing cyclodextrins are now commercially used to produce low calorie beer with 1% more alcohol content.
(c) All the genes of an entirely new biosynthetic pathway may be transferred to generate new products. For example, two genes are involved in the conversion of acetyl-CoA to polyhydroxybutyrate (PHB), which is used to produce biodegradable plastic. The two genes were transferred into E. coli from Alcaligenes eutrophus. Transgenic E. coli. under appropriate conditions, accumulate PHB to upto 50% of their dry weight.
(d) Several gene transfers have enhanced growth rates of the organisms, reduced their nutrient requirements and enabled their growth to higher cell densities. For example, transfer of the gene encoding glutamate dehydrogenase from E. coli to glutamate synthase (GOGAT) deficient mutants of Methylophilus methylotrophus increased the efficiency of carbon conversion from 4% to 7%.
M. methylotrophus is used to produce microbial biomass from methanol; this biomass is used as animal feed. Glutamate dehydrogenase utilizes ammonium to produce glutamic acid but uses little energy, while glutamate synthase pathway (in which GOGAT is involved) uses up one additional ATP molecule for each molecule of glutamic acid produced.
(e) In some cases, conversion of an intermediate product to the end product is slow due to a low activity of the rate-limiting enzyme. In such cases, the activity of rate-limiting enzyme can be increased by increasing its dosage.
For example, in case of C. acremonium, the enzyme (encoded by the gene cefEF) that converts penicillin N intermediate in the cephalosporin C biosynthesis is rate-limiting. The dosage of cefEF was increased leading to a 15% higher cephalosporin C yield. (The penicillin content was reduced by a factor of 15).
The above listed examples are only a fraction of the possibilities offered by the recombinant-DNA technology. The powers of this technology shall increase with the refinements of techniques for genetic modification of industrially important microorganisms and the knowledge of various details of their metabolic pathways.