丙酮酸羧化酶对酿酒酵母积累琥珀酸的作用探究  

Effects of pyruvate carboxylase on succinic acid accumulation in Saccharomyces cerevisiae

作　　者：陈园园 程慧 李宁娜 苏珂 李佳雨[1,3] 夏华龙 徐国强 张梁 CHEN Yuanyuan;CHENG Hui;LI Ningna;SU Ke;LI Jiayu;XIA Hualong;XU Guoqiang;ZHANG Liang((National Engineering Laboratory for Cereal Fermentation Technology(Jiangnan University),Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education(Jiangnan University),Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,china)

机构地区：[1]粮食发酵工艺与技术国家工程实验室(江南大学),江苏无锡214122 [2]工业生物技术教育部重点实验室(江南大学),江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2019年第13期38-44,共7页Food and Fermentation Industries

基　　金：国家自然科学基金重大项目(21390204);国家自然科学基金青年基金(31300027);中国博士后基金一等资助(2014M550266);工业生物技术教育部重点实验室开放课题(KLIB-KF201606)

摘　　要：为探究丙酮酸羧化酶及微量元素Ca^2+和生物素对酿酒酵母积累琥珀酸的作用,敲除了琥珀酸脱氢酶编码基因(SDH2)并过量表达来自米根霉的丙酮酸羧化酶基因(RoPYC)。琥珀酸产量由(0.011±0.002)g/L提高至(0.841±0.020)g/L,较表达前提高了75.45%。随后,外源添加不同浓度Ca^2+和生物素考察其对琥珀酸发酵的影响,结果发现,Ca^2+的添加促进了菌体的生长但不利于琥珀酸的积累,而外源添加浓度为16、32、64、96μg/L生物素时,琥珀酸产量分别提高75.04%、84.26%、69.28%、66.79%。其中生物素质量浓度为32μg/L时,琥珀酸产量达到最大为(0.964±0.02)g/L,且丙酮酸羧化酶(pyruvate carboxylase,PC)酶活提高14.42%,说明PC酶活的提高促进了酿酒酵母中琥珀酸的积累。该研究为解决酿酒酵母琥珀酸合成过程中前体碳代谢流不足问题提供了新的解决思路。This study was constructed to explore the effects of pyruvate carboxylase as well as trace elements Ca^2+and biotin on succinic acid accumulation in Saccharomyces cerevisiae.The succinate dehydrogenase gene(SDH2)of S.cerevisiae was knocked out and pyruvate carboxylase gene(RoPYC)from Rhizopus oryzae was over-expressed.The results showed that the production of succinic acid increased by 75.45%from(0.011±0.002)g/L to(0.841±0.020)g/L.Moreover,it was found that Ca^2+promoted the growth of the cells but did not have effects on succinic acid accumulation.In comparison,16,32,64,and 96μg/L biotin enhanced the production of succinic acid by 75.04%,84.26%,69.28%,and 66.79%,respectively.Additionally,succinic acid production reached a maximum of(0.964±0.02)g/L when adding 32μg/L biotin,and the activity of pyruvate carboxylase increased by 14.42%.This indicated that the increment of pyruvate carboxylase activity promoted the accumulation of succinic acid in S.cerevisiae.This study provides a new solution to the insufficient carbon flow in succinic acid synthesis in S.cerevisiae.

关 键 词：丙酮酸羧化酶 代谢工程 琥珀酸 酿酒酵母 

分 类 号：TS261.11[轻工技术与工程—发酵工程]