提高光滑球拟酵母乙酰辅酶A水平促进α－酮戊二酸合成  

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

作　　者：梁楠[1] 王淼[2] 刘立明[1] 堵国成[1] 陈坚[1] 

机构地区：[1]工业生物技术教育部重点实验室,江南大学食品学院无锡214122 [2]江南大学食品学院,无锡214122

出　　处：《微生物学报》2008年第7期874-878,共5页Acta Microbiologica Sinica

基　　金：国家杰出青年基金(20625619);国家“863计划”(2006AA02Z201);国家自然科学基金(30670066,20706025);国家“973项目”(2007CB71403)~~

摘　　要：【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(1.20U/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0.94mmol/(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、α-酮戊二酸产量和Cα-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4g/L乙酸,使乙酰辅酶A浓度、α-酮戊二酸产量和Cα-KG/Cpyr是出发菌株WSH-IP303的4.55、2.47和3.75倍,α-酮戊二酸浓度达到17.8g/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α-酮戊二酸。[Objective] This study aimed at increasing the α-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, α-ketoglutaric acid, and the value of C α-Kc/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 α-ketoglutaric acid concentration reached 17.8 g/L by increasing the availability of acetyl-CoA and this strategy may provide an alternative approach to enhance metabolite production in yeast.

关 键 词：光滑球拟酵母 乙酰辅酶A合成酶 乙酰辅酶A 

分 类 号：Q93[生物学—微生物学]