State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China.
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China; School of Life Science, Linyi University, Linyi 276000, China.
Metab Eng. 2017 Jul;42:126-133. doi: 10.1016/j.ymben.2017.06.007. Epub 2017 Jun 13.
Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7g/L succinic acid with a yield of 0.53g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.
解脂耶氏酵母因其能够积累柠檬酸循环中间产物和耐受酸性 pH 值而被认为是生产琥珀酸的潜在候选菌株。先前，通过缺失琥珀酸脱氢酶亚基编码基因 Ylsdh5 获得了一种琥珀酸生产菌株。然而，副产物乙酸盐的积累限制了琥珀酸产量的进一步提高。同时，额外的 pH 调节步骤增加了工业应用中的下游成本。在本研究中，我们首次发现，线粒体中乙酰辅酶 A 向琥珀酸的 CoA 转移反应而不是 SDH 阴性 Y. lipolytica 中的丙酮酸脱羧反应导致乙酸盐溢出。CoA 转移酶基因 Ylach 的缺失消除了乙酸盐的形成，提高了琥珀酸的产量和细胞生长。然后，我们分析了过表达氧化三羧酸、还原羧化和乙醛酸旁路的关键酶对琥珀酸产量和副产物形成的影响。过表达来自酿酒酵母的磷酸烯醇丙酮酸羧激酶（ScPCK）和内源性琥珀酰辅酶 A 合酶β亚基（YlSCS2）的最佳菌株提高了琥珀酸产量 4.3 倍。在分批补料发酵中，该菌株在无需 pH 控制的情况下以 0.53g/g 甘油的得率生产了 110.7g/L 的琥珀酸。这是迄今为止，在使用限定培养基的情况下，在低 pH 值下由酵母实现的最高琥珀酸产量。本研究不仅揭示了 SDH 阴性 Y. lipolytica 中乙酸盐溢出的机制，而且还报道了一种具有巨大工业前景的高效琥珀酸生产菌株的开发。
