Regulatory mutants

Two types of mutants have been used for amino add overproduction auxotrophic and regulatory mutants. In some cases, mutant strains have been further improved through DNA-recombination. 

There are two possible sites that are genetically inactivated in regulatory mutants ... 

Selection of these regulatory mutants is often done by using toxic analogues of amino adds for example p-fluoro-DL-phenylalanine is an analogue of phenylalanine. Mutants that have no feedback inhibition or repression to the amino add are also resistant to the analogue amino add. They are therefore selected for and can be used to overproduce the amino add. Some amino add analogues function as false co-repressors, false feedback inhibitors or inhibit the incorporation of foe amino acid into foe protein. 

The best amino add producers are organisms that are both auxotrophic and regulatory mutants. 

Unlike auxotrophic mutants, regulatory mutants can be grown in inexpensive, complex media and they do not require careful control of growth conditions. 

Regulatory mutants improve the rate of L-phenylalanine overproduction by... 

Since auxotrophic mutants and regulatory mutants are widely used in the overproduction of amino adds, this can be a severe problem. In nature, mutation always takes place but this takes some time. However, in fermentation many generations are produced in a relatively short period of time and the chances of back mutation are enhanced. 

Regulatory mutants are not subject to feedback inhibition, even by L-phenylalanine itself. 

Hydrogenase activities in regulatory mutants of R. capsulatus affected in transcription of hydrogenase genes compared to the wild-type BIO... 

Figure 3.15 Hydrogenase synthesis in regulatory mutants of R. eutropha. Cells were grown on agar plates containing glycerol as the carbon source either in the presence or in the absence H2.The cell material was transferred to filter paper and a triphenyl tetra-zolium chloride-based hydrogenase activity staining was performed. Dark colour reflects activity of the MBH.

Supek V, Gamulin S, Delic V. (1985) Enhancement of bacitracin biosynthesis by branched-chain amino acids in a regulatory mutant of Bacillus licheniformis. Folia Microbiol 30 342-348. 

Pospisil S, Peterkova M, Krumphanzl V, Vanek Z. (1984) Regulatory mutants of Streptomyces cinnamonensis producing monensin A. FEMS Microbiol Lett 24 209-213. 

The terminal amino acids are under strict metabolic control. Some act as feedback inhibitors or repressors. Their synthesis is in equilibrium with metabolic requirement. This equilibrium position prevents their accumulation and hence the yield of these compounds is low. By changing the growth requirement (environmental stimulus) or by genetic manipulation, mutants could be found with limited or removed feedback inhibitors and repressors, e.g. auxotrophic and regulatory mutants 49). This needed a better understanding of biosynthesis and regulation of amino acid production. By selection of these mutants it became possible to alter microbial metabolism which led to the accumulation of the desired amino acids. 

A fermentation process for producing lysine was made possible by using mutants of Corynebacterium glutamicum or Brevibacterium flavum. Both auxotrophic and regulatory mutants have been obtained for overproduction of lysine. Figure 30.19 shows the biosynthetic... 

Komatsubara S, Kisumi M, Murata K et al (1978) Threonine production by regulatory mutants of Serratia marcescens. Appl Environ Microbiol 35 834—840... 

Greenberg, M., Goldwasser, P., and Henry, S., 1982a, Characterization of a yeast regulatory mutant constitutive for inositol-1-phosphate synthase. Mol. Gen. Genet. 186 157-163. 

Swede, M.J., 1994, Isolation and characterization of novel regulatory mutants of phospholipid biosynthesis in Saccharomyces cerevisiae. Department of Biological Sciences, Carnegie Mellon University. 

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