Aspartokinase mutants

S-(2-Aminoethyl)-L-cysteine (AEC), H2N-CH2-CH2-S-CH2-CH(NH2)-COOH, a lysine analog, acts as a false feedback inhibitor on aspartokinase, which produces aspartylphosphate from aspartate. The inhibitor simulates, for aspartokinase, the absence of lysine and threonine, and as a consequence the AEC insensitive mutant is no longer inhibited by lysine and threonine. The result was a yield increase from 0 to 16 g L 1. 

E. coli The mutant aspartokinase III encoded by lysC was used to enhance L-threonine production A 30.9% increase in L-threonine production 14.4 Ogawa-Miyata et al. [57]... 

Removal of negative regulations can also enhance L-threonine production. By removing L-lysine-mediated feedback inhibition of aspartokinase III encoded by the lysC gene, L-threonine production could be increased by 30.9% (11.0-14.4 g L 1) in E. coli mutant strain [57]. 

The pathway of biosynthesis of L-lysine and L-threonine in Corynebacterium glutamicum is shown in Fig. 1. The first step, the formation of phosphoaspartate from aspartate, is catalyzed by aspertokinase and this enzyme is susceptible to the concerted feedback inhibition by L-lysine and L-threonine. The auxotrophic mutant of homoserine (or threonine plus methionine), lacking homoserine dehydrogenase, was constructed and found to produce L-lysine in the culture medium. Second, the mutants which show the threonine or methionine sensitive phenotype caused by the mutation on homoserine dehydrogenase (low activity) was also found to produce appreciable amounts of L-lysine in the culture medium. Furthermore, a lysine analogue (S-aminoethylcysteine) resistant mutant was obtained as an L-lysine producer and in this strain aspartokinase was insensitive to the feedback inhibition. 

Attempts were therefore made to increase carbon flow into the threonine pathway by enhancing the activity of the lysine-sensi-tlve aspartokinase. This was accomplished by selecting nitroso-guanidine-lnduced mutants resistant to aminoethylcysteine (61). 

Kato C, Kurihara T, Kobashi N, Yamane H, Nishiyama M (2004) Conversion of feedback regulation in aspartate kinase by domain exchange. Biochem Bioph Res Co 316 802-808 Kim YH, Park JS, Cho JY, Cho KM, Park YH, Lee J (2004) Proteomic response analysis of a threonine-overproducing mutant of Escherichia coli. Biochem J 381 823-829 Klaffl S, Eikmanns BJ (2010) Genetic and functional analysis of the soluble oxaloacetate decarboxylase from Corynebacterium glutamicum. J Bacterid 192 2604-2612 Komatsubara S, Kisumi M, Murata K, Chibata 1 (1978) Threonine production by regulatory mutants of Serratia marcescens. Appl Environ Microbiol 35 834-840 Kotaka M, Ren J, Lockyer M, Hawkins AR, Stammers DK (2006) Structures of R- and T-state Escherichia coli aspartokinase 111 mechanisms of the allosteric transition and inhibition by lysine. J Biol Chem 281 31544-31552... 

The intermediate of L-lysine biosynthesis, L-aspartyl-semialdehyde, is also a precursor for the biosynthesis of L-threonine, L-isoleucine, and L-methionine. Homoserine dehydrogenase catalyses NADPH-dependent reduction of L-aspartyl-semialdehyde to L-homoserine. Flux from L-aspartyl-semialdehyde toward L-homoserine could be reduced by introducing alleles for less active homoserine dehydrogenase variants. In addition, L-lysine production increased as the prevailing threonine concentrations in such horn mutants were too low for feedback inhibition of aspartokinase [67, 68]. 

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