Examples Of The Use Of Auxotrophs For The Production Of Primary Metabolites

Many auxotrophic mutants have been produced from C. glutamicum for the synthesis of both amino acids and nucleotide related compounds. C. glutamicum is a biotin-requiring organism which will produce glutamic acid under biotin-limited conditions but it is important to remember that mutants of this organism, employed for the production of other amino acids, must be supplied with levels of biotin optimum for growth. Biotin-limited conditions will result in these mutants producing glutamate and not the desired amino acid.

Auxotrophic mutants of C. glutamicum have been used for the production of lysine. The control of the production of the aspartate family of amino acids in C-glutamicum is shown in Fig. 3.14. Aspartokinase, the

Aspartate

Aspartyl phosphate

Aspartyl phosphate

Lysine Biosynthesis Glutamicum

Isoleucine

» Biosynthetic route

-a Feedback repression

Isoleucine

» Biosynthetic route

-a Feedback repression

Fig. 3.14. The control of the aspartate family of amino acids in C. glutamicum.

first enzyme in the pathway, is controlled by the concerted feedback inhibition of lysine and threonine. Homoserine dehydrogenase is subject to feedback inhibition by threonine and repression by methionine. The first enzyme in the route from aspartate semialdehyde to lysine is not subject to feedback control. Thus, the control system found in C. glutamicum is a relatively simple one. Nakayama et al. (1961) selected a homoserine auxotroph of C. glutamicum, by the penicillin selection and replica plating method, which produced lysine in a medium containing a low level of homoserine, or threonine plus methionine. The mutant lacked homoserine dehydrogenase which allowed as-partic semi-aldehyde to be converted solely to lysine and the resulting lack of threonine removed the concerted feedback inhibition of aspartokinase. Ki-noshita and Nakayama (1978) quoted the homoserine auxotroph, C. glutamicum 901, as producing 44 g dm'3 lysine.

The control of the production of arginine in C. glutamicum is shown in Fig. 3.15. The major control of the pathway is the feedback inhibition of the second enzyme in the sequence, acetylglutamic acid phospho-rylating enzyme, although the first enzyme may also be subject to regulation.

Kinoshita et al. (1957b) isolated a citrulline requiring auxotroph of C. glutamicum which would accumulate ornithine at a molar yield of 36% from glucose, in the presence of limiting arginine and excess biotin. The mutant lacked the enzyme converting ornithine to citrulline which resulted in the cessation of arginine synthesis and, therefore, the removal of the control of the pathway.

Inosine monophosphate has been shown to demonstrate flavour-enhancing qualities and is produced commercially by the chemical phosphorylation of ino-

Glutamate

/V-acetyl glutamate

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