提高光滑球拟酵母乙酰辅酶A水平促进a-酮戊二酸合成

【目的】为了了解光滑球拟酵母中乙酰辅酶A含量对其碳代谢及其通量的影响。【方法】将来源于酿酒酵母中编码乙酰辅酶A合成酶ACS2基因过量表达于发酵法生产丙酮酸的生产菌株Torulopsis glabrata中，获得了一株乙酰辅酶A合成酶活性提高9.2倍(1.20 U/mg protein)的重组菌T. glabrata ACS2-1。【结果】与出发菌株WSH-IP303相比，重组菌T. glabrata ACS2-1：(1)能以乙酸为唯一碳源在胞内积累0.94 mmol/(L·g DCW)的乙酰辅酶A；(2)以葡萄糖为唯一碳源时胞内乙酰辅酶A浓度、a-酮戊二酸产量和Ca-KG/Cpyr是出发菌株WSH-IP303 的3.22、2.05和2.52倍；(3)在葡萄糖培养基中添加4 g/L乙酸，使乙酰辅酶A浓度、a-酮戊二酸产量和Ca-KG/Cpyr是出发菌株WSH-IP303的4.55、2.47和3.75倍，a-酮戊二酸浓度达到17.8 g/L。【结论】这一结果表明，改变细胞内关键辅因子的浓度能使碳代谢流的流向与通量发生改变，从积累丙酮酸转向过量积累a-酮戊二酸。

Enhancing a-ketoglutaric acid production in Torulopsis glabrata: increase of acetyl-CoA availability

Objective] This study aimed at increasing the a-ketoglutaric acid production of a multi-vitamin auxotrophic yeast Torulopsis glabrata, by increasing the availability of acetyl-CoA. Methods] For this, we expressed ACS2 encoding acetyl-CoA synthase from Saccharomyces cerevisiae in the pyruvate producer Torulopsis glabrata WSH-IP303. Results] Compared with that of the parent strain, the acetyl-CoA synthase activity of the mutant ACS2-1 increased about 920% and the mutant could use acetate as the sole carbon source for growth (2.6 g/L dry cell weight). When growing with glucose, the acetyl-CoA concentration, a-ketoglutaric acid, and the value of C a-KG/Cpyr were 222%, 105% and 152% higher than those of the parent strain WSH-IP303, respectively. The addition of 4 g/L acetate to the culture broth of mutant ACS-1 led to a significant increase of these values to 355%, 147% and 275%, respectively, compared with that of the parent strain WSH-IP303. Conclusion] The a-ketoglutaric acid concentration reached 17.8 g/L by increasing the availability of ace-tyl-CoA and this strategy may provide an alternative approach to enhance metabolite production in yeast.