Pines O, Shemesh S, Battat E, Goldberg I
Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Appl Microbiol Biotechnol. 1997 Aug;48(2):248-55. doi: 10.1007/s002530051046.
Saccharomyces cerevisiae accumulates L-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a 6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in L-malic acid accumulation in the production medium. The high apparent Km of MDH2 for L-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function in the enzyme and differs from the previously published Km of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be a limiting factor, thus providing a system for further metabolic engineering of L-malic acid production. The overexpression of MDH2 activity also causes an evaluation in the accumulation of fumaric acid and citric acid. Accumulation of fumaric acid is presumably caused by high intracellular L-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing possibility that cytosolic L-malic acid is a direct precursor of citric acid in yeast.
酿酒酵母通过一条从丙酮酸开始的胞质途径积累L-苹果酸，该途径涉及丙酮酸羧化酶和苹果酸脱氢酶。在本研究中，对苹果酸脱氢酶在胞质途径中的作用进行了研究。在强诱导型GAL10或组成型PGK启动子下过表达胞质苹果酸脱氢酶（MDH2），会使生长培养基和生产培养基中的胞质MDH活性提高6至16倍，在生产培养基中L-苹果酸积累量最多可提高3.7倍。MDH2对L-苹果酸的高表观Km值（11.8 mM）表明该酶对这种酸的亲和力较低，这与该酶的胞质功能一致，且与先前发表的线粒体酶（MDH1，0.28 mM）的Km值不同。在MDH2过表达的条件下，丙酮酸羧化酶似乎是一个限制因素，从而为L-苹果酸生产的进一步代谢工程提供了一个系统。MDH2活性的过表达还导致富马酸和柠檬酸积累量的评估。富马酸的积累可能是由于细胞内L-苹果酸浓度较高以及胞质富马酸酶的活性所致。柠檬酸的积累可能暗示了一种有趣的可能性，即胞质L-苹果酸是酵母中柠檬酸的直接前体。
