烟酸转磷酸核糖激酶和丙酮酸羧化酶共表达对大肠杆菌BA002产丁二酸的影响  

Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002

作　　者：曹伟佳[1] 苟冬梅[1] 梁丽亚[1] 刘嵘明[1] 陈可泉[1] 马江锋[1,2] 姜岷[1] 

机构地区：[1]南京工业大学生物与制药工程学院材料化学工程与国家重点实验室,江苏南京211816 [2]中国石化扬子石油化工有限公司南京研究院,江苏南京210048

出　　处：《生物工程学报》2013年第12期1855-1859,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.21076105);国家重点基础研究发展计划(973计划)(No.2009CB724701);江苏高校优势学科建设工程项目;国家高技术研究发展计划(863计划)(No.2011AA02A203);新世纪优秀人才支持计划(No.NCET-12-0732)资助~~

摘　　要：大肠杆菌BA002是敲除了乳酸脱氢酶的编码基因(ldhA)和丙酮酸-甲酸裂解酶的编码基因(pflB)的工程菌。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。pncB是烟酸转磷酸核糖激酶(NAPRTase)的编码基因,通过过量表达pncB基因能够提高NAD(H)总量与维持合适的NADH/NAD+,从而恢复了厌氧条件下重组菌E.coli BA014(BA002/pTrc99a-pncB)的生长和产丁二酸的性能。然而,BA014在厌氧发酵过程中有大量丙酮酸积累,为进一步提高菌株的丁二酸生产能力,减少副产物丙酮酸的生成,共表达NAPRTase和来自于乳酸乳球菌NZ9000中丙酮酸羧化酶(PYC)的编码基因pyc,构建了重组菌E.coli BA016(BA002/pTrc99a-pncB-pyc)。3 L发酵罐结果表明,BA016发酵112 h后,共消耗了35.00 g/L的葡萄糖。发酵结束时,菌体OD600为4.64,产生了25.09 g/L丁二酸。通过共表达pncB和pyc基因,使BA016的丙酮酸积累进一步降低,丁二酸产量进一步提高。Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD＋. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase （NAPRTase） encoded by the pncB gene, a rate-limiting enzyme of NAD（H） synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase （PYC） of Lactococcus Iactis subsp, cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that 00600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.

关 键 词：烟酸转磷酸核糖激酶 丙酮酸羧化酶 丁二酸 NADH NAD+ 厌氧发酵 

分 类 号：TQ921.4[轻工技术与工程—发酵工程]