一种基于途径分析提高丙酮酸产量的育种策略  

Application of a metabolic-pathway-analysis based breeding strategy enhances the production of pyruvate by Torulopsis glabrata

作　　者：刘立明[1] 李寅[1] 陈坚[1] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,无锡214036

出　　处：《微生物学报》2005年第1期77-80,共4页Acta Microbiologica Sinica

基　　金：江苏省自然科学基金 (BK2 0 0 2 0 72 );江苏省高等学校研究生创新计划~~

摘　　要：为进一步提高光滑球拟酵母发酵生产丙酮酸的水平 ,在途径分析的基础上提出了一种组成型降低丙酮酸脱酸酶、但增强乙酰辅酶A合成酶活性的育种策略。通过亚硝基胍诱变 ,获得 1株乙酸需求型突变株CCTCCM2 0 2 0 19,在外加乙酸的培养基中表现出高于出发株 2 1%的丙酮酸生产能力和良好的遗传稳定性。检测突变株CCTCCM2 0 2 0 19中丙酮酸代谢相关酶的活性发现 :(1)丙酮酸脱羧酶活性降低了 4 0 % ;(2 )外加乙酸与否的条件下 ,乙酰辅酶A合成酶的活性分别提高了 10 3 5 %和 5 7 4 % ;(3)添加乙酸和突变对丙酮酸羧化酶、丙酮酸脱氢酶系、乙醇脱氢酶和乙醛脱氢酶的活性没有显著影响。在含有乙酸的培养基中突变株细胞干重比出发株高 2 1 7% ,可能是因为乙酰辅酶A合成酶活性的提高 ,补充了因丙酮酸脱羧酶活性降低而引起的胞质乙酰辅酶A短缺。在 7L罐中含有 6g L乙酸钠的培养基中发酵 6 2h ,丙酮酸产量达到 6 8 7g L ,对葡萄糖的产率为 0 6 5 1g g。To further enhance the pyruvate productivity by multi-vitamin auxotrophic yeast Torulopsis glabrata, a breeding strategy aiming at decreasing the activity of pyruvate decarboxylase but increasing the activity of acetyl-CoA synthetase was developed based on analysis of pyruvate-related metabolic pathways. Nitrosoguanidine mutagenized cells of T. glabrata WSH-IP303 were screened for mutants that require acetate for complete growth on glucose minimum medium. A mutant, T. glabrata CCTCC M202019, produced pyruvate 21% higher than that of the parent strain and was genetically stable in flask cultures, was selected as a working strain. To elucidate the metabolic changes that led to the increase of pyruvate production, the activities of enzymes that involved in pyruvate-related metabolic pathways of the mutant and the parent strain were determined. Enzymatic analysis revealed that, compared with the parent strain WSH-IP303, the activity of pyruvate decarboxylase of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase of the latter increased by 103.5% or 57.4%, respectively, in the presence or absence of acetate. When 6g/L sodium acetate was added to the medium, pyruvate production by the mutant strain CCTCC M202019 reached 68.7g/L at 62h (yield on glucose, 0.651g/g) in fermentations performed in a 7-L jar fermentor, indicating the shortage of cytosolic acetyl-CoA resulted from the disruption of pyruvate decarboxylase was properly compensated by the increase of the activity of acetyl-CoA synthetase.

关 键 词：光滑球拟酵母 丙酮酸 丙酮酸脱羧酶 乙酰辅酶A合成酶 育种策略 

分 类 号：TQ920[轻工技术与工程—发酵工程]