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