School of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China,
Biotechnol Lett. 2013 Dec;35(12):2091-8. doi: 10.1007/s10529-013-1302-7. Epub 2013 Jul 27.
Malic enzyme (EC 1.1.1.40) converts L-malate to pyruvate and CO2 providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the malic enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD(+) as the primary cofactor. Km values for NAD(+) and NADP(+) were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and α-ketoglutaric acid (>5 mM) were inhibitory while succinate (5-15 mM) increased NADP(+)- but not NAD(+)-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP(+)-dependent malic enzyme, the malic enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that malic enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.
苹果酸酶（EC 1.1.1.40）将 L-苹果酸转化为丙酮酸和 CO2，为代谢提供 NADPH，特别是在产油微生物中进行脂质生物合成。然而，其在产油酵母解脂耶氏酵母中的作用尚不清楚。我们从解脂耶氏酵母中克隆了苹果酸酶基因（YALI0E18634g），并将其插入 pET28a 中，在大肠杆菌中表达并纯化了重组蛋白（YlME）。YlME 使用 NAD(+) 作为主要辅助因子。NAD(+) 和 NADP(+) 的 Km 值分别为 0.63 和 3.9 mM。柠檬酸、异柠檬酸和α-酮戊二酸（>5 mM）具有抑制作用，而琥珀酸（5-15 mM）增加了 NADP(+)-但不增加 NAD(+)-依赖性活性。为了确定通过提供来自 NADP(+)-依赖性苹果酸酶的额外 NADPH 是否可以增加解脂耶氏酵母中的脂肪酸生物合成，我们在解脂耶氏酵母中表达了来自产油真菌白僵菌的苹果酸酶基因（mce2）。脂质含量或脂肪酸谱没有发生显著变化，这表明苹果酸酶不是解脂耶氏酵母中脂质积累的 NADPH 的主要来源。
