Biochemical Engineering Fundamentals, Part 1 - Bailey J., Ollis D. 1989

Molecular Genetics and Regulatory Systems
Industrial Applications of Research on Microbial Genetics and Mutant Populations
Mutants with Modified Regulatory Systems

More industrially advantageous strains of microorganisms have also been obtained by modifying regulatory systems at the enzyme and (or) Gene level using other genetic transformations and Selection. Mutants of this type can be employed to enhance the yields of metabolic products and Enzymes.

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FIG. 6.20. In a normal Cell, AMP and GMP inhibit the nucleotide Biosynthesis pathway via feedback inhibition at several stages (a). In a mutant unable to synthesize AMP and GMP, the concentrations of these substances can be maintained at a sufficiently low level by adjusting the medium composition, thereby increasing the yield of inosine and hypoxanthine (b). (Aiba S., Humphrey A., Millis N., Biochemical Engineering and Equipment. — M.: Pishchevaya Promyshlennost, 1975.)

To achieve the overproduction of a metabolite that exhibits inhibitory and (or) repressor properties in its own biosynthetic pathway, it is necessary to screen for mutant organisms whose allosteric enzymes and operons are insensitive to the presence of the metabolite. Such mutants can frequently be isolated using antimetabolites—toxic analogs of the studied metabolites. Normal Cells fail to grow in an antimetabolite-containing medium because the antimetabolite suppresses or inhibits The biosynthesis of the essential metabolite, while being unable to perform the Functions of the non-synthesized metabolite or replace it in downstream metabolic pathways. Conversely, the biosynthetic profile and reaction rates of strains with impaired feedback regulation systems remain unaltered in the presence of the antimetabolite; therefore, such strains grow normally even in a medium containing it. Table 6.6 lists several microbial products whose yields can be increased via this approach.

Table 6.6. Metabolic products and corresponding antimetabolites whose application in microbial selection allows the isolation of more productive mutants^a

^a Demain A. L., Overproduction of Microbial Metabolites due to Alteration in Regulation, in Advances in Biochemical Engineering, vol. 1, Ghose T. K., Fiechter A. (eds.), p. 113, Springer-Verlag, New York, 1971.

Resistance to antimetabolites is often driven by various alterations in regulatory systems, such as the partial or complete restoration of The activity of enzymes whose catalytic properties are suppressed in the normal biosynthetic pathway. Specifically, antimetabolites can also be used to screen for mutants with unusually high concentrations of biosynthetic enzymes. This approach may prove particularly useful, for instance, when developing production processes for enzymes subsequently applied in other in vitro biosynthetic processes.

Constitutive mutants are characterized by the fact that the biosynthesis of inducible or repressible enzymes does not depend on the presence or absence of an inducer or repressor. By utilizing constitutive mutants, the yields of the following Enzymes can be increased: ß-galactosidase, catalase, Phosphatases, proteases, homoserine dehydrogenase, invertase, histidase, penicillinase, and amidase.

In the preceding Structure/133.html">Discussion, we focused primarily on various approaches to increasing the yield of target substances. These very same molecular biological approaches can serve as a basis for exploring biosynthetic routes to derivatives of normal metabolites, as well as entirely novel compounds that may find Applications in pharmacology and other fields. This topic is briefly reviewed in [4–6]. A practically significant example of such a process is the biosynthesis of the tetracycline antibiotic 6-demethyltetracycline by a mutant strain of Streptomyces aureofaciens. 6-Demethyltetracycline is more stable in acidic environments than conventional tetracycline and is therefore one of the principal tetracycline Antibiotics produced on an industrial scale.