Regulation and bypassing of regulatory mechanisms for the over-production of Secondary metabolites

Regulation and bypassing of regulatory mechanisms for the over-production of Secondary metabolites

Regulation of secondary metabolites production

The formation of secondary metabolites is regulated by nutrients, growth rate, feedback control, enzyme inactivation, and enzyme induction.  Regulation is influenced by unique low molecular mass compounds, transfer RNA, sigma factors and gene products formed during post-exponential development.

The synthesis of secondary metabolites is often coded by clustered genes on chromosomal DNA and less frequently on plasmid DNA. Unlike primary metabolism, the pathways of secondary metabolism are still not much understood. Secondary metabolism is brought on by exhaustion of a nutrient, biosynthesis or addition of an inducer, and/or by a growth rate decrease. These events generate signals which affect a cascade of regulatory events resulting in chemical differentiation or secondary metabolism. The signal is often a low molecular weight inducer which acts by negative control.  It binds to and inactivate a regulatory protein (repressor protein/receptor protein) which normally prevents secondary metabolism during rapid growth and nutrient sufficiency.

There are several levels of hierarchy for regulation of secondary metabolite production and morphogenesis.

Approaches for overproduction of secondary metabolites

1. Modification of microbial response (Elicitation, Quorum Sensing)

A. Elicitors

Environmental abiotic and biotic stress factors have been proved to effect variety of responses in

microbes. Elicitors, as stress factors, induce or enhance the biosynthesis of secondary metabolites.

They are classified based on their nature and origin: physical or chemical, biotic or abiotic.

Abiotic stress (abiotic elicitors) imposed by pH improves antibiotic production by Streptomyces spp.

The effect of carbohydrate biotic elicitors (oligosaccharides, oligomannuronate, oligoguluronate and mannan- oligosaccharides) on variety of fungal systems: Penicillium spp.,Ganoderma spp., Corylopsis spp. And bacterial cultures: Streptomyces spp., Bacillus spp. for production of antibiotics, enzymes, pigments were investigated.

B. Quorum Sensing

Quorum sensing is the communication between cells through the release of chemical signals when cell density reaches a threshold concentration (critical mass. Under these conditions, they sense the presence of other microbes.

A number of physiological activities of microbes such as symbiosis, competence, conjugation, sporulation, biofilm formation, virulence, motility and the production of various secondary metabolites are regulated through the quorum-sensing. There is great potential for the use of this communication process for industrial exploitation. There is possibility of overproduction of fungal metabolites in response to the supplementation by variety of quorum sensing molecules.

Precursors often stimulate production of secondary metabolites either by increasing the amount of a limiting precursor, by inducing a biosynthetic enzyme or both. These are usually amino acids but other small molecules also function as inducers.

2. Genetic engineering (Strain Improvement)

Genetic engineering methods are divided into two: Classical genetic methods and Molecular genetic improvement methods.

A. Classical Genetic Methods

I. mutation and random selection

II. mutation and rational selection

III. Genetic recombination methods

Mutation and Random Selection: Relied on mutation, followed by random screening, then careful secondary screening tests are performed and new improved mutants are selected. Physical mutagens such as UV-light or chemical mutagens are used in these methods. Advantages of Classical genetic methods are simplicity, no need to sophisticated equipment, minimal specialized technical manipulation, effectiveness. The drawback is it is labour intensive.

Mutation and Rational Selection (Directed Selection Techniques): This involves selection of a particular characteristic of the desired genotype, different from the one of final interest, but easier to detect. Design of these methods requires some basic understanding of the product metabolism and pathway regulation.  For example, addition of a toxic precursor of penicillin to the agar medium of penicillin producing microorganisms prevents the growth of sensitive strains and only resistant mutants with more penicillin production will be grown.

Genetic Recombination Methods: Recombination by protoplast fusion between related species of fungi results in high productivity.

B. Molecular Genetic Improvement Methods

Require knowledge and tools to perform molecular genetic improvement and that include, identification of biosynthetic pathway, adequate vectors and effective transformation protocols. The main strategies being used are as follows:

·         Amplification of secondary metabolite Biosynthetic Genes (Targeted duplication or amplification of SM production gene)

·         Inactivation of Competing Pathways

·         Disruption or Amplification of Regulatory Genes

·         Manipulation of Secretory Mechanisms

·         Expression of a Convenient Heterologous Protein

·         Combinatorial Biosynthesis

3. Metabolic Engineering

Metabolic engineering can be defined as purposeful modification of cellular metabolism using recombinant DNA and other molecular biological techniques. Many drugs and drug precursors found in natural organisms are rather difficult to synthesize chemically and to extract in large amounts. Metabolic engineering is playing an increasingly important role in the production of these drugs and drug precursors. This is typically achieved by establishing new metabolic pathways leading to the product formation, and enforcing or removing the existing metabolic pathways toward enhanced product formation.

Examples of successful examples of metabolic engineering include the efficient production of L-valine, L-threonine, lycopene, antimalarial drug precursor, and benzylisoquinoline alkaloids

4. Ribosome Engineering

Researchers found a dramatic activation of antibiotic production by a certain ribosomal mutation and bacterial gene expression may be changed dramatically by modulating the ribosomal proteins or rRNA, eventually leading to activation of inactive genes.

 

References

1.      Overproduction Strategies for Microbial Secondary Metabolites: A Review, Nafiseh Davatia and Mohammad. B Habibi Najafib, International Journal of Life Science and Pharma Research, Vol 3, Issue 1, 2013

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

 

Regulation and bypassing of regulatory mechanisms for the over-production of Primary metabolites

Regulation and bypassing of regulatory mechanisms for the over-production of Primary metabolites 

Metabolites are the intermediates and products of metabolism, are typically characterized by small molecules with various functions.

Metabolites can be categorized into Primary metabolites and secondary metabolites.

Primary metabolites are microbial products made during the exponential phase of growth and the synthesis of primary metabolites are integral part of the normal growth process. Examples of primary metabolites are

·         Intermediates and end-products of anabolic metabolism, used by the cell as building blocks for essential macromolecules (e.g. amino acids, nucleotides) or are converted to coenzymes (e.g. vitamins)

·         Other primary metabolites (e.g. citric acid, acetic acid and ethanol) result from catabolic metabolism, not used for building cellular constituents but related to energy production and substrate utilization

Industrially, the most important primary metabolites are amino acids, nucleotides, vitamins, solvents, ethanol and organic acids. These are made by a diverse range of bacteria and fungi and have numerous uses in the food, chemical and nutraceutical industries.

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 modified to provide conditions that would fully exploit the increased potential of the culture.  Thus, the process of strain improvement involves the continual genetic modification of the culture, followed by reappraisals of its cultural requirements for maximum industrial production of the particular primary or secondary metabolite.

 Regulation of primary metabolism

Microbial metabolism is a conservative process that usually does not produce already available compounds and does not overproduce required molecules. Their metabolic machinery is highly regulated so that only necessary enzymes at correct amount are made.

Transcription is the principal site for control and is dependent on transcription factors. Transcription factors are proteins which bind near or at promoters, to activate or repress transcription initiation. To initiate transcription in bacteria, RNA polymerase associate with sigma factor.  This is small protein that direct RNA polymerase to promoter sequences. In most bacteria, there are different sigma factors, which allow the cell to carry out basal gene expression, exponential growth and to respond to environmental signals. Escherichia coli makes seven sigma factors whereas Bacillus subtilis makes seventeen. There are also anti-sigma factors which bind to and inhibit sigma factor function. There are anti-anti-sigma factors also, which are antagonists of anti-sigma factors. A wide range of cellular processes are regulated by anti-sigma factors, including bacteriophage growth, sporulation, stress response, flagellar biosynthesis, pigment production, ion transport and virulence expression.

The primary control of gene expression in eukaryotes is also at the level of transcription and is exerted by transcription factors. While prokaryotic transcription factors bind close to the gene to be transcribed, eukaryotic transcription factors often bind hundreds or thousands of base pairs upstream of the gene. Upstream of eukaryotic genes is the TATA box, which binds transcription factor. Transcription factors include (i) helix–turn–helix structures, (ii) zinc fingers, (iii) leucine zippers, (iv) helix–loop– helix structures and (v) high-mobility groups. After binding to DNA, the factors interact with other factors or with RNA polymerase itself to modulate transcription either in the positive direction (transcription activation) or in the negative direction (transcription repression).

Regulatory mechanisms involved in the biosynthesis of primary metabolites

1. Induction.

This is a control mechanism by which a substrate (or a compound structurally similar to the substrate, or a metabolically related compound) ‘turns on’ the synthesis of enzymes, which are usually involved in the degradation of the substrate. Enzymes that are synthesized as a result of genes being turned on are called inducible enzymes and the chemical that activates gene transcription is called the inducer.

Inducible enzymes are produced only in response to the presence of their substrate and are produced only when needed. In this way, the cell does not waste energy synthesizing unneeded enzymes. The inducer molecule combines with a repressor at the DNA level and thereby prevents the blocking of an operator by the repressor, leading to the transcription of the gene and translation of the messenger RNA encoding the enzyme. Although most inducers are substrates, sometimes products can function as inducers. As examples, malto-dextrins can induce amylase, fatty acids induce lipase, urocanic acid induces histidase, and galacturonic acid induces polygalacturonase. Some coenzymes induce enzymes, as in thiamine induction of pyruvate decarboxylase. Substrate analogues that are not attacked by the enzyme (‘gratuitous inducers’) are often excellent inducers of enzyme synthesis.

The most thoroughly studied inducible enzyme system is that for lactose hydrolysis in E. coli - lac operon -negative control of protein synthesis.  Positive regulation of transcription occurs in E. coli for utilization of L-rhamnose, maltose and arabinose and galactose utilization in Saccharomyces cerevisiae.

2. Carbon source regulation. Like enzyme induction, carbon source regulation (carbon catabolite repression (CCR)) is one of the conservative mechanisms against wasting a cell’s protein-synthesizing machinery, and operates when more than one utilizable substrate is present in the environment.

The cell produces enzymes to catabolize the most rapidly assimilable carbon source while synthesis of enzymes utilizing other substrates is repressed until the primary substrate is exhausted. The repressed enzymes are usually inducible. Carbon catabolite repression is a phenomenon usually caused by glucose, but in different organisms, other carbon sources can also cause repression. An example of this occurs in Pseudomonas aeruginosa, where citrate is the preferred carbon source over glucose.

Adenylate cyclase, cAMP and cAMP–CRP complex are involved in Carbon catabolite repression.  It is present in E. coli, Bacillus species, P. aeruginosa, Arthrobacter crystallopoietes, Rhizobium meliloti and anaerobic bacteria, e.g. Bacteroides fragilis. etc.

In molds such as A. nidulans the gene for carbon catabolic repression is creA.

3. Nitrogen source regulation. Nitrogen can be assimilated from inorganic or organic sources. Its assimilation from inorganic sources requires reduction to ammonia, followed by incorporation into intracellular metabolites. The distribution of nitrogen among various pathways involves specific regulatory mechanisms, such as end-product inhibition or end-product-mediated transcriptional control. Nitrogen source regulation (NSR) is known by many other names such as nitrogen metabolite repression, nitrogen catabolite repression and ammonia repression. Enzymes typically under such control are proteases, amidases, ureases and those that degrade amino acids.

The ability to assimilate particular inorganic or organic nitrogen sources depends on the particular organism. Organic nitrogen sources are usually monomeric units (e.g. amino acids or nucleobases) or compounds derived from them (e.g. agmatine or putrescine). Ammonia usually supports the fastest growth rate and is therefore considered the preferred nitrogen source for E. coli.

4. Phosphorus source regulation. In natural environments, inorganic phosphorus is commonly the major growth-limiting nutrient. Thus, biological systems have evolved a variety of responses to modulate their phosphorus requirement or to optimize its utilization. In E. coli, over 30 genes are part of the phosphate regulon (Pho regulon) and are transcriptionally activated by phosphorylated PhoB when the cell finds itself in low phosphate. These genes encode proteins involved in uptake and utilization of phosphorus compounds.

Nucleases and phosphatases are usually repressed by phosphate in fungi. In addition, phosphate represses proteases, isocitrate lyase, fructose diphosphate aldolase, NADP isocitrate dehydrogenase and malate dehydrogenase in Neurospora. Phosphate also suppresses the production of riboflavin by Eremothecium ashbyii

5. Sulfur source regulation. Sulfatases are regulated by sulfate and sulfur amino acids. In addition to a variety of nutrients used to maintain continuous growth, bacteria require a source of sulfur. The use of sulfur is controlled by transcriptional regulatory proteins.

6. Feedback regulation. Feedback regulation is the most important mechanism responsible for regulation of the enzymes involved in biosynthesis of amino acids, nucleotides and vitamins. This category of regulation functions at two levels: enzyme action (feedback inhibition) and enzyme synthesis (feedback repression and attenuation).

In feedback inhibition, the final metabolite of a pathway, when present in sufficient quantities, inhibits the action of the first enzyme of the pathway to prevent further synthesis of intermediates and products of that pathway.

Feedback repression involves the turning off of enzyme synthesis when sufficient amounts of the product have been made and it starts to accumulate. The end-product of the pathway acts as a co-repressor. It activates the aporepressor and active repressor will be formed which binds to the operator to prevent transcription and hence prevents enzyme synthesis.

Attenuation (transcription termination control) involves charged tRNA (tRNA with corresponding amino acid) for transcription termination. Many of the amino acid biosynthetic pathways are regulated not by the amino acids themselves but by their charged tRNA molecules. In the presence of an excess of charged tRNA, transcription is initiated but terminated between the operator and the first structural gene.  Attenuation control certain bacterial amino acid biosynthetic operons, e.g. threonine, isoleucine, valine, leucine, phenylalanine, histidine.

The tryptophan operon is regulated by both repression and attenuation.

7. Additional types of regulation. Other types of regulation include metabolic interlock, stringent control and regulatory inactivation.

Approaches for overproduction of primary metabolites

1 Mutation and screening or selection

Organisms for industrial production of primary metabolites were initially developed by mutation followed by selection or screening. Such efforts often start with multiple mutations in organisms having some capacity to make the desired product.

These mutations involve the release of feedback controls and enhance the formation of pathway precursors and intermediates. This approach of strain improvement successfully produced organisms that make industrially significant concentrations of primary metabolites. However, there are some problems such as  (i) the necessity of screening large numbers of mutants and (ii) the chances of the producing strain to get lose of their synthetic potential.

2. Genetic engineering

Modern genetic engineering techniques could be used to develop strains overproducing primary metabolites. This is particularly valuable in organisms with complex regulatory systems. Production of a particular primary metabolite may be increased by

1.      increasing the number of copies of structural genes coding for these enzymes by genetic engineering - by incorporating the biosynthetic genes in vitro into a plasmid which, when placed in a cell by genetic transformation, will replicate into multiple copies

2.      increasing the frequency of transcription - involves constructing a hybrid plasmid in vitro, which contains the structural genes of the biosynthetic enzymes but lacks the regulatory sequences. The structural genes are placed next to an efficiently and frequently read promoter and operator

One of the major problems in using such techniques is the difficulty in maintaining the plasmids during fermentation. Plasmid instability in the absence of selective pressure leads to a dilution of the plasmid in the population and loss of the desired phenotype. One solution is to use antibiotic pressure during fermentation so that only organism resistant to the antibiotic due to the presence of a plasmid-borne resistance gene can survive. Plasmid stabilization can also be accomplished by cloning in E. coli carrying a temperature-sensitive mutation. At the non-permissive temperature, growth will be dependent on the plasmid carrying the particular gene for temperature stability which also contains our product coding gene .

3. Novel genetic technologies ‘Genome-based strain reconstruction’ achieves the construction of a superior strain which contains mutations crucial to hyperproduction only. This approach was used to improve lysine production

 

 References

1.      Metabolic regulation and overproduction of primary metabolites, Sergio Sanchez and Arnold L. Demain, Microbial Biotechnology (2008) 1(4), 283–319

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

 

 

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