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