Strain selection and improvement of industrial micro-organisms

Strain selection and improvement of industrial micro-organisms

Natural isolates usually produce commercially important products in very low concentrations.  Increased yields may be achieved by optimizing the culture medium and growth conditions, but this approach will be limited by the organism's maximum ability to synthesize the product. The potential productivity of the organism is controlled by its genome and, therefore, the genome must be modified to increase the potential yield. The cultural requirements of the modified organism would then be examined to provide conditions that would fully exploit the increased potential of the culture, while further attempts are made to beneficially change the genome of the already improved strain. Thus, the process of strain improvement involves the continual genetic modification of the culture, followed by reappraisals of its cultural requirements.

Genetic modification may be achieved by selecting natural variants, by selecting induced mutants or by selecting recombinants.

In a Strain Improvement Program, in general, economic is the major motivation.  Metabolite concentrations produced by the wild types are too low for economical processes. For cost effective processes improved strain should be attained which

·                     Do not show catabolite repression

·                     Have permeability alterations to improve product export

·                     require shorter fermentation times

·                     do not produce undesirable products

·                     have reduced oxygen needs

·                     cause lower viscosity of the culture so that oxygenation is less of a problem

·                     exhibit decreased foaming during fermentation

·                     have tolerance to high concentrations of carbon or nitrogen sources

The success of strain improvement depends greatly on the target product. Raising gene increase the product, (products involving the activity of one or a few genes), such as enzymes.  This may be beneficial if the fermentation product is cell biomass or a primary metabolite.  However, with secondary metabolites, which are frequently the end result of complex, highly regulated biosynthetic processes, a variety of changes in the genome may be necessary to permit the selection of high-yielding strains.

The selection of natural variants

It is not possible to rely on natural variants for improvements in productivity since there is a small probability of a genetic change occurring each time a cell divides and when it is considered that a microbial culture will undergo a vast number of such divisions it is not surprising that the culture will become more heterogeneous. The heterogeneity of some cultures can present serious problems of yield degeneration because the variants are usually inferior producers compared with the original culture.

Thus selecting induced mutants and selecting recombinants are usually done.

The selection of induced mutants synthesizing improved levels of primary metabolites

Isolation of mutants producing products whose biosynthesis and control have been sufficiently understood which enables to prepare 'blueprints' of the desirable mutants.

The levels of microbial metabolites are controlled by a variety of mechanisms, such that end products are synthesized in amounts not greater than those required for growth. However, the ideal industrial micro-organism should produce amounts far greater than those required for growth and

1.                  The organism may be modified such that the end products which control the key enzymes of the pathway are lost from the cell due to some abnormality in the permeability of the cell membrane.

2.                  The organism may be modified such that it does not produce the end products which control the key enzymes of the pathway.

3.                  The organism may be modified such that it does not recognize the presence of inhibiting or repressing levels of the normal control metabolites.

a. Isolation of analogue resistant mutants.

An analogue is a compound which is very similar in structure with compound and analogues mimic the compound in binding but the pathway cannot be completed.

Resistant mutants may be isolated by exposing the survivors of a mutation treatment to a suitable concentration of the analogue in growth medium and purifying any colonies which develop.  Gradient plate technique could be used.

Analogue is a compound which is very similar in structure to another compound.  Generally analogues of aminoacids, vitamins, nucleotides, etc are growth inhibitory or highly toxic since they impair with the normal metabolism by mimicking their natural molecule and altering the control mechanisms.  Analogue resistant mutants are mutants which does not identify or recognize the product or its structural analogue as a feed back inhibitor and as a result the organism will continue to produce the product in high levels without any feedback inhibition.  Isolation of analogue resistant mutants may be done by using the gradient plate technique  The gradient plate technique allows a gradual, proportional increase of drug concentration in the agar medium.  Here the Resistant mutants are isolated by exposing the survivors of a mutation treatment to a suitable concentration of the analogue in a growth medium and purifying the colonies which develop. In brief, the organism after the mutation treatment will be exposed to a range of concentrations of the toxic analogue.  Colonies which develop in the presence of the analogue will be resistant mutants.  We can expose the organisms to a range of analogue concentrations on a single plate by gradient plate technique.  Molten agar medium, containing the analogue will be poured into a slightly slanted petri dish and allowed to set at an angle. After the agar has set, a layer of medium not containing the analogue is added and allowed to set with the plate level. The analogue will diffuse into the upper layer giving a concentration gradient across the plate.  When the survivors of a mutation treatment spread over the surface of the plate and incubated, resistant mutants can be detected as isolated colonies appearing in the region of high concentration of the analogue while a zone of confluent growth will be there in the region having low concentration of analogue.  The isolated colonies from the high concentration zone are the analogue resistant mutants.  These mutant will have improved productivity due to their inability to recognize the presence of the end product as a feed back inhibitor.

b. Isolation of revertants

Auxotrophic mutants may revert to the phenotype of the mutant 'parent', but the reversion may result in loss of the regulatory properties of particular enzyme.

Auxotrophic mutants (which have lost the potential of producing a particular metabolite) may revert to the phenotype of the mutant 'parent' (that is it reverted back to parent type, now capable of producing the particular metabolite) and may regain the ability to produce a particular product.  But sometimes during this reversion mutation, the enzyme of the revertant may lose its ability to be controlled through feedback inhibition by the product.   

The isolation of induced mutants producing improved yields of secondary metabolites - where directed selection is difficult to apply

Depend on the random selection of the survivors of mutagen exposure.  "Hit or miss methods that require brute force, persistence and skill in the art of microbiology".  Involves subjecting a population of the micro-organism to a mutation treatment and then screening a proportion of the survivors of the treatment for improved productivity.

Decisions are to be made on

(i) How many colonies from the survivors of a mutation treatment should be isolated for testing?

(ij) Which colonies should be isolated?

Miniaturized techniques are used to grow the survivors of the mutation treatment either in a very low volume of liquid medium or on solidified (agar) medium. If the product is an antibiotic, the agar-grown colonies may be overlayed with an indicator organism sensitive to the antibiotic produced, allowing assay to be done in situ. The level of antibiotic is assessed by the degree of inhibition of the overlayed indicator.

A more directed selection approach has been adopted for the improvement of secondary metabolite producers such as isolation of auxotrophs, revertants and analogue-resistant mutants.

Isolation of auxotrophic mutants

Supplementation of that particular nutrient may enhance the secondary metabolite productivity

Isolation of revertant mutants

A mutant may revert to the phenotype of its 'parent', but the genotype of the revertant may not, necessarily, be the same as the original 'parent'. Some revertant auxotrophs have been demonstrated to accumulate secondary metabolites.

(i) The isolation of revertants of mutants auxotrophic for primary metabolites which may influence the production of a secondary metabolite.

(ij) The reversion of mutants which have lost the ability to produce the secondary metabolite

The isolation of analogue resistant mutants

Mutants may be isolated which are resistant to the analogues of primary metabolic precursors of the secondary metabolite, or resistant to the feedback effects of the secondary metabolite or resistant to the toxic effects of the secondary metabolite or resistant to the toxic effects of a compound due to the production of the secondary metabolite.

The use of recombination systems for the improvement of industrial micro-organisms for primary and secondary metabolite

"any process which helps to generate new combinations of genes that were originally present in different individuals".

·   The parasexual cycle

·   Protoplast fusion techniques

·   Recombinant DNA techniques

The parasexual cycle

Many industrially important fungi do not possess a sexual stage and therefore it is difficult to achieve recombination in these organisms. However nuclear fusion and gene segregation could take place in the absence of sexual organs. The process was termed the parasexual cycle.

In order for parasexual recombination to take place in an imperfect fungus, nuclear fusion must occur between unlike nuclei in the vegetative hyphae of the organism.  Thus, recombination may be achieved only in an organism in which at least two different types of nuclei coexist, i.e. a heterokaryon. The major components of the parasexual cycle are the establishment of a heterokaryon, vegetative nuclear fusion and mitotic crossing over or haploidization resulting in the formation of a diploid or haploid recombinant.

Disadvantages

·         No recombination may occur

·         Induction of heterokaryons is a difficult process

·         Diploids produced by the parasexual cycle are frequently unstable

Protoplast fusion techniques

Protoplasts are cells devoid of their cell walls and may be prepared by subjecting cells to the action of wall degrading enzymes in isotonic solutions. Protoplasts may regenerate their cell walls and are then capable of growth as normal cells. Cell fusion, followed by nuclear fusion, may occur between protoplasts of strains which would otherwise not fuse and the resulting fused protoplast may regenerate a cell wall and grow as a normal cell.

Fusion of fungal protoplasts appears to be an excellent technique to obtain heterokaryons between strains

Recombinant DNA techniques

The transfer of DNA between different species of bacteria has been achieved.  Thus, genetic material derived from one species may be incorporated into another where it may be expressed.

·         A 'vector' DNA molecule (plasmid or phage) capable of entering the host cell and replicating within it.

·         A method of splicing foreign genetic information into the vector.

·         A method of introducing the vector/foreign DNA recombinants into the host cell and selecting for their presence.

·         A method of assaying for the 'foreign' gene product of choice from the population of recombinants created.

The production of heterologous proteins

The first commercial heterologous protein to be produced was human growth hormone (hGH) which is used to treat hypopituitary dwarfism and, prior to its manufacture by fermentation, was extracted from the brains of human cadavers.

The use of recombinant DNA technology for the improvement or increase of native microbial products

Chromosomal gene is inserted into a plasmid and the plasmid incorporated into the original strain and maintained at a high copy number

The improvement of industrial strains by modifying properties other than the yield of product

Some examples of the characteristics which may be important in this context are

·      Selection of stable strains

·      Selection of strains resistant to infection

·      Selection of non-foaming strains

·      Selection of strains which are resistant to components in the medium

·      The selection of morphologically favourable strains

·      The selection of strains which are tolerant of low oxygen tension

·      The elimination of undesirable products from a production strain

·      The development of strains producing new fermentation products such as semisynthetic penicillins

 

 

References

1.                  Principles of fermentation technology, PF Stanbury, A Whittakker, SJ Hall, 1995, Butterworth-Heinemann publications

2.                  Industrial microbiology -Casida