高产光学纯(R,R)-2,3-丁二醇工程菌株构建与发酵优化  

Construction and optimization of an engineered strain for efficiently producing enantiomerically pure(R,R)-2,3-butanediol

作　　者：黄艳燕[1] 陈先锐 彭龙云 叶柳健 李检秀[1] 蒙健宗[2,3] 张云开 谢能中[1,3] Yanyan Huang;Xianrui Chen;Longyun Peng;Liujian Ye;Jianxiu Li;Jianzong Meng;Yunkai Zhang;Nengzhong Xie(College of Life Science and Technology, Guangxi University, Nanning 530004, China;National Engineering Research Center for Non - food Biorefinery, State Key Laboratory of Non - food Biomass and Enzyme Technology, Guangxi Biomass Industrialization Engineering Institute, Guangxi Key Laboratory of Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China;Nanning Sinozyme Biotechnology Company Limited, Nanning 530007, China)

机构地区：[1]广西科学院国家非粮生物质能源工程技术研究中心非粮生物质酶解国家重点实验室广西生物质产业化工程院广西生物炼制重点实验室,广西南宁530007 [2]广西大学生命科学与技术学院,广西南宁530004 [3]南宁中诺生物工程有限责任公司,广西南宁530007

出　　处：《生物技术》2018年第2期178-184,共7页Biotechnology

基　　金：广西科技计划项目(“生物质高效转化光学纯(R,R)-2,3-丁二醇关键技术引进与合作研究”,No.桂科合14125008-2-22);国家自然科学基金项目(“基于基因组学的多粘类芽孢杆菌合成(2R,3R)-2,3-丁二醇研究”,No.21466007)

摘　　要：[目的]构建能够专一性合成光学纯(R,R)-2,3-丁二醇的大肠杆菌工程菌,并进行发酵条件优化。[方法]将来源于多粘芽孢杆菌的(R,R)-2,3-丁二醇脱氢酶基因bdh,来源于阴沟肠杆菌的α-乙酰乳酸合成酶基因bud B和α-乙酰乳酸脱羧酶基因bud A与表达载体p Tr C99A连接,导入大肠杆菌中构建人工合成途径。筛选最适的培养基和发酵条件,提高(R,R)-2,3-丁二醇的产量、产率和得率。[结果]获得高效合成(R,R)-2,3-丁二醇的工程菌株GXASB,筛选到最适碳源及其浓度为120 g/L木薯淀粉,最适pH为6.5,最适接种量为10%,在发酵罐中进行同步糖化法发酵,(R,R)-2,3-丁二醇产量达到105.28 g/L,光学纯为99.1%,得率为0.47 g/g,生产强度为1.95 g/(L·h)。[结论]在大肠杆菌中表达基因簇bud B-bud A-bdh能够专一性合成光学纯(R,R)-2,3-丁二醇,经优化发酵条件后,能够显著提高(R,R)-2,3-丁二醇的合成效率。同时工程菌能够利用非粮原料木薯淀粉高效生产(R,R)-2,3-丁二醇,补料发酵产量达到105.28 g/L,为使用廉价原料工业化生产(R,R)-2,3-丁二醇提供参考。[Objective]To construct an Escherichia coli engineered strain for enantiomerically pure（R,R）-2,3-butanediol production and to optimize the fermentation conditions.[Methods]A synthetic pathway consisting of bud B and bud A from Enterobacter cloacae,and（R,R）-2,3-bdh from Paenibacillus polymyxa,was constructed in E.coli.The single factor comparative experiments were adopted to optimize the fermentation culture and conditions to improve the（R,R）-2,3-butanediol titers,yields and productivities.[Results]An engineered strain GXASB was gained for（R,R）-2,3-butanediol biosynthesis.And the optimized carbon source was 120 g/L cassava starch,the optimized pH and the inoculums was 6.5 and 10% respectively.Furthermore,（R,R）-2,3-butanediol concentration of fed-batch fermentation reached 105.28 g/L（Enantio-purity99.1%） within 54 h with the yield of 0.47 g/g,the productivity of 1.95 g/（L·h）.[Conclusion]Expression of gene cluster bud B-bud A-bdh in E.coli engineered strain could generate enantiomerically pure（R,R）-2,3-butanediol specifically.And optimization of the fermentation culture and conditions led to a higher yield of（R,R）-2,3-butanediol.Non-food material cassava starch showed a lot of promise to become a low cost raw material for the industrial production of（R,R）-2,3-butanediol in the future.

关 键 词：(R R)-2 3-丁二醇 人工合成途径 合成生物学 大肠杆菌基因工程菌 非粮原料 发酵条件优化 

分 类 号：TQ923[轻工技术与工程—发酵工程]