| 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 |
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| 引用本文： | 曹伟佳,苟冬梅,梁丽亚,刘嵘明,陈可泉,马江锋,姜岷.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[J].生物工程学报,2013,29(12):1855-1859. |
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| 作者姓名： | 曹伟佳 苟冬梅 梁丽亚 刘嵘明 陈可泉 马江锋 姜岷 |
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| 作者单位： | 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;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;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;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;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;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；中国石化扬子石油化工有限公司南京研究院，江苏 南京 210048;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 |
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| 基金项目： | 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。 |
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| 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。
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| 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 |
| 收稿时间： | 4/9/2013 12:00:00 AM |
Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002 |
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| Weijia Cao,Dongmei Gou,Liya Liang,Rongming Liu,Kequan Chen,Jiangfeng Ma and Min Jiang.Effect of co-expression of nicotinic acid phosphoribosyl transferase and pyruvate carboxylase on succinic acid production in Escherichia coli BA002[J].Chinese Journal of Biotechnology,2013,29(12):1855-1859. |
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| Authors: | Weijia Cao Dongmei Gou Liya Liang Rongming Liu Kequan Chen Jiangfeng Ma and Min Jiang |
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| Institution: | State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China;State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China;State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China;State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China;State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China;State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China; Nanjing Research Institute of Sinopec Yangzi Petrochemical Company Limited, Nanjing 210048, Jiangsu, China;State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China |
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| Abstract: | 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 lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 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. |
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| Keywords: | nicotinic acid phosphoribosyl transferase pyruvate carboxylase succinc acid NADH/NAD+ anaerobic fermentation |
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