Toward biotechnological production of adipic acid and precursors from biorenewables.

(1985). α-Aminoadipate Pathway for the Biosynthesis of Lysine in Lower Eukaryotes. CRC Critical Reviews in Microbiology: Vol. 12, No. 2, pp. 131-151.

alpha-Aminoadipate pathway for the biosynthesis of lysine in lower eukaryotes.

Genetics of antibiotic production.

The biochemistry of the 13 steps involved in the conversion of α-ketoglutarate and acetylCoA to α-ketosuberate, a precursor to the coenzymes coenzyme B (7-mercapto heptanoylthreonine phosphate) and

Alpha-keto acid chain elongation reactions involved in the biosynthesis of coenzyme B (7-mercaptoheptanoyl threonine phosphate) in methanogenic Archaea.

Homocitrate synthase in the first enzyme of the lysine biosynthetic pathway. It is feedback regulated by L-lysine. Lysine decreases the biosynthesis of penicillin (determined by the incorporation of [14C]valine into penicillin) by inhibiting and repressing homocitrate synthase, thereby depriving the cell of alpha-aminoadipic acid, a precursor of penicillin. Lysine feedback inhibited in vivo the biosynthesis and excretion of homocitrate by a lysine auxotroph, L2, blocked in the lysine pathway after homocitrate. Neither penicillin nor 6-aminopenicillanic acid exerted any effect at the homocitrate synthase level. The molecular mechanism of lysine feedback regulation in Penicillium chrysogenum involved both inhibition of homocitrate synthase activity and repression of its synthesis. In vitro studies indicated that L-lysine feedback inhibits and represses homocitrate synthase both in low- and high-penicillin-producing strains. Inhibition of homocitrate synthase activity by lysine was observed in cells in which protein synthesis was arrested with cycloheximide. Maximum homocitrate synthase activity in cultures of P. chrysogenum AS-P-78 was found at 48 h, coinciding with the phase of high rate of penicillin biosynthesis.

Inhibition and repression of homocitrate synthase by lysine in Penicillium chrysogenum.

Homocitric acid accumulation by a lysine-requiring yeast mutant.

Homocitric acid (β-hydroxy-β-carboxyadipic acid; HC) is accumulated by a lysine-requiring yeast mutant when grown in a chemically defined medium, supplemented with limited amounts of lysine. A study of the formation of HC in relation to the depletion of lysine from the growth medium indicates that HC accumulated only when the concentration of lysine was low. The enzymatic formation of HC from α-ketoglutarate plus acetyl-coenzyme A in cell-free extracts of the same organism was also inhibited by lysine. The inhibitory effect of lysine on the formation of HC in both whole cells and cell-free extracts is indicative of the functional existence of a feedback control mechanism in the pathway for lysine biosynthesis in yeast.

Control of Lysine Biosynthesis in Yeast by a Feedback Mechanism

Cell-free extracts of Acetobacter suboxydans were prepared which were capable of condensing α-ketoisovalerate with 14C-labeled acetyl-coenzyme A to yield 14C-labeled α-isopropylmalate. The product of the reaction was isolated by paper and column chromatography and was characterized by recrystallization with synthetic α-isopropylmalic acid to constant specific radioactivity. The formation of α-isopropylmalate by extracts of A. suboxydans plus the ability of the organism to grow in a simple glucose-glycerol medium containing glutamic acid as the only amino acid indicate that the pathway for leucine biosynthesis shown to exist in yeast and Salmonella typhimurium also occurs in A. suboxydans. As a comparison, the condensation of oxalacetate and (14C) acetyl-coenzyme A to yield (14C) citric acid was shown, by similar means, to occur in A. suboxydans. This is of interest since the existence of this classical condensing enzyme has hitherto not been demonstrated in this organism. This reaction was further demonstrated in cell-free extracts of A. suboxydans by means of a spectrophotometric assay at 232 mμ which measured the cleavage of the carbon-sulfur bond of acetyl-coenzyme A in the presence of oxalacetate. Comparison of the specific activities of crude cell-free extracts indicated a much more extensive occurrence of this reaction in yeast than in A. suboxydans.

Biosynthesis of α-Isopropylmalic and Citric Acids in Acetobacter suboxydans

Evidence indicated an accumulation of l-malic acid in the synthetic growth medium of both lysine auxotrophs and wild-type yeast. Malic acid was isolated from growth experiments and was identified by paper and column chromatography, by comparison of infrared spectra with authentic l-malic acid, as well as enzymatically, as a substrate of malic dehydrogenase. Malic acid was accumulated when glucose was used as a carbon source. This did not occur when sodium lactate was the carbon source. Malic, succinic, and citric acids, and a few other as yet unidentified organic acids, were accumulated in the culture supernatant fluid when yeast was grown in a chemically defined medium. The accumulation of these acids had no obvious relation to the pathway for lysine biosynthesis.

Michael E. Maragoudakis

Papers

Homocitric acid accumulation by a lysine-requiring yeast mutant.

Control of Lysine Biosynthesis in Yeast by a Feedback Mechanism

Biosynthesis of α-Isopropylmalic and Citric Acids in Acetobacter suboxydans

Identification of Malic Acid from Yeast