In this article we will discuss about the improvement of microbial strains for better production of products.
1. Methods of Strain Improvement:
A mutant requiring oleic acid for neomycin formation by Streptomyces fradiae showed a decrease in the intracellular level of neomycin precursors in the mutant. On the other hand supersensitive mutants of β-lactam antibiotics are another example.
Recent approaches towards strain improvement are given below:
(i) Role of Plasmid:
Plasmid genes are involved in antibiotic production in Streptomyces spp. Although, plasmids are involved in genetic characteristics on curing experiments. Involvement of plasmids in biosynthesis of aureothricin and kasugamycin in Str. kasuaensis was demonstrated more than decades ago by Okanishi (1970).
The genetic study using Str. venezuelae ISP 5230 a chloramphenicol (CM) producer contains most of the structural genes for the CM biosynthetic steps treated between met and ilu on the chromosome and the plasmid played role in increasing CM production. A linear plasmid like DNA (pSLA2) of 11.2 × 106 dalton molecular weight from Streptomyces sp. produced antibiotics.
(ii) Protoplast Fusion:
Protoplast fusion is one of the useful techniques for obtaining hybrids or recombinants of different microorganism strains. Various studies have been carried out by using protoplast fusion in Streptomyces, Saccharomyces, and fungi.
Protoplast formation in Sterptomyces was first reported by Okanishi and his team in the year 1966. Further, they have worked on formation stabilization and regeneration of protoplast of Str. griseus and Str. venezuelae. Fusion of yeast protoplasts has been reported with Sacchromyces cerevisiae. Technique for protoplast fusion in Brevibacterium flavum, has been used for strain improvement.
Screening after major subjection of a parent strain to physical or chemical mutagen greatly increased the probability of finding improved strain.
(a) Major mutations:
It involves the selection of mutants with a pronounced change in a biochemical character of practical interest. Such variants are commonly used in genetic studies and are generally low mutants’.
They are isolated routinely from population surviving after prolonged exposure to a mutagen, for example, selection of non-pigmented Penicillium chrysogenum strains with high penicillin production. The initial strain of Sterptomyces griseus (a streptomycin producing organism) synthesized the small amount of streptomycin but its variant was isolated which produced greater amount of streptomycin.
For further improvement it is also necessary to study the biosynthetic pathways which contribute to the identification of precursors as in case of a modified tetracycline synthesized by a mutant strain of Str. aureofacies.
The molecule got changed at the C-5 position and was almost devoid of antibiotic activity. Another mutant strain S-604 synthesized 6-dimethyl tetracycline, a new antibiotic, not elaborated by the parent strains, proved to have several advantages. Today it is one of leading commercial forms of tetracycline.
(b) Minor mutations:
It plays a dominant role in strains improvement. By definition such mutation affects only the amount of product synthesized. Such variants are usually phenotypically similar to the parent, with rapid and abundant mycelial and conidial development.
A 10 to 15% increase in conidial population exposed to moderate doses of a mutagen, obtained after repeated isolation of minor (positive) variants and using each succeeding strain for further mutation and selection Such increases have also been obtained by repeated selection without the introduction of mutagen In this case, the population to be tested must be large and assay for the desired product also must be accurate and specific.
This technique fetched importance in improving P. chrysogenum. For example, Wisconsin series were the famous Q-176 culture with significantly improved antibiotic titres, and strains BL3-D10, which does not produce the characteristic and trouble some chrysogenin pigment. All further mutant selections over the next decade were derived from Q-176.
2. Mutation Concept for Strain Development:
Strains selected as obvious variants after exposure to mutagen are usually inferior in their capacity for accumulation of antibiotic. Improvements are extremely few and their selection and evaluation is extremely important.
Mutagen dose is important. Mutants sought for major mutation rates are best isolated from populations surviving prolonged doses of mutagen, whereas variants for increased productivity are generally isolated from population surviving intermediates dose level.
Strains with enhanced altered morphology, etc. may be inherently better producers but may require considerable fermentation development. Step wise selection implies small increment in productivity, and the probability of getting hyper producing strains decreases.
Variant strains may require special propogation and preservation procedures and actual production gains depend also on stability and reliability of performance.
Though, strains may prove better in their productivity at laboratory scale, there is no guarantee that enhanced productivity will occur in production fermenters. The long term pilot plant studies are often necessary before any enhanced strains potential can be realized in actual production.
3. Isolation of Mutant Classes and Their Use in Microbial Processes:
(i) Localized Mutagenesis and Computation:
Localized mutagenesis affecting the small selected regions of the chromosomes, offers a promising new approach. Mutation programmes can be directed to maximize mutations in any marked area on the chromosome, specially the areas known to affect the formation of end products. Isolation of strains in unknown loci linked to the revertant site can be done by a heterokaryon method or by the use of temperature sensitive mutants.
(ii) Sexual and Parasexual Processes:
In fungi, the vegetative mycelium is haploid and can be propogated almost indefinitely by serial transfer of hyphal fragments and can also be propogated by asexual spores/conidia.
Two strains of opposite mating types (A or B) are required to initiate the sexual cycle and allow to mate by mixing the conidia of mating type A with mycelia on appropriate media. After a period of nuclear division and migration, fusion between A and B nuclei takes place. Each fused nucleus (diploid) undergoes meiosis to form four haploids, which divide mitotically into eight nuclei contained in ascus.
Few of the industrially important fungi form heterokaryon in which rare diploid nuclei result from the fusion of two haploid nuclei. This process is called Parasexuality. Although, recombination in fewer fragments in the parasexual cycle compared to the meiotic process it can occur by mitotic crossing over or by other mechanism.
The importance of mitotic crossing over or recombination is that it makes possible genetic analysis and controlled breeding in organisms with no sexual cycle. Stram improvement through parasexual cycle has been reported in P. chrysogenum and in one study; a homozygous diploid representing parent was an efficient producer of penicillin V.