Osamu Tosaka

Pathway and Regulation of Lysine Biosynthesis in Brevibacterium lactofermentum

The pathway and regulation of lysine biosynthesis in Brevibacterium lactofermentum were investigated. The biosynthetic pathway of l-lysine in Brevibacterium was the same as that in Escherichia coli, since biosynthetic enzymes, which were aspartokinase [EC 2.7.2.4], dihydrodipicolinate (DDP)* synthetase, DDP reductase and N-acetyl-e-keto-α-aminopimelate (AKAP) synthetase, could be detected in B. lactofermentum.AKAP synthetase was measured with 1Δ-piperideine-2,6-dicarboxylate, which was prepared from l-diaminopimelate by l-amino acid oxidase, and either acetyl-CoA or succinyl-CoA could be served as an acyl donor for this reaction.Effect of amino acids on these enzymes was estimated. Aspartokinase was inhibited about 41, 45 and 82% by single or simultaneous addition of l-threonine and l-lysine at 1 mm, respectively, whereas the addition of other amino acids did not cause significant inhibitory effect.In contrast with DDP synthetase from E. coli, B. lactofermentum enzyme was not affected by l-lysine.DDP redu...

Production of L-Lysine by Fluoropyruvate-sensitive Mutants of Brevibacterium lactofermentum

Better producers of l-lysine were obtained by derivation of fluoropyruvate(FP)-sensitive mutants from Brevibacterium lactofermentum AJ3990. The coexistence of FP and excess biotin synergistically stimulated l-lysine formation by washed cells. FP inhibited 50% of growth and pyruvate dehydrogenase (PDH) activity of AJ3990 at 0.04 mm and 1 mm, respectively. Therefore, the synergistic effect of FP and excess biotin seems to be due to the optimization of the PDH/pyruvate carboxylase activity ratio in l-lysine biosynthesis. This was confirmed by the derivation of FP-sensitive mutants which have the optimal level of PDH activity for l-lysine production. The best producer, AJ11204, had about 27% PDH activity as compared with the parental strain and accumulated 70 g of l-lysine per liter with a conversion yield of 50% from glucose in the presence of excess biotin.

Effect of Biotin Levels on l-Lysine Formation in Brevibacterium lactofermentum

The relationship between l-lysine production and biotin levels in culture medium was investigated in Brevibacterium lactofermentum. Strain AJ3799 accumulated 34 mg/ml and 42 mg/ml of l-lysine HCl in the presence of 50 μg/liter and 500 μg/Iiter, respectively, of biotin in culture medium. This increase in l-lysine production was observed with various kinds of lysine producers and also with resting cells grown in biotin poor medium. Biotin functioned as an effector only when glucose or pyruvate was used as a substrate. And this effect was accompanied with reduction of carbon dioxide liberation, as well as decreased accumulation of l-alanine, l-valine and l-leucine. On the other hand, penicillin and some surfactants had no effect on the function of biotin. These data suggest a significant new role for biotin in Brev. lactofermentum.