过量表达钝齿棒杆菌柠檬酸合酶编码基因prpC2对L-精氨酸合成的影响

doi:10.13523/j.cb.20150307

中国生物工程杂志

2015, Vol. 35

Issue (3): 49-55 DOI: 10.13523/j.cb.20150307

研究报告

过量表达钝齿棒杆菌柠檬酸合酶编码基因prpC2对L-精氨酸合成的影响

房战¹, 徐美娟¹, 饶志明¹, 满在伟¹, 许正宏², 耿燕², 陆茂林³

1. 江南大学工业生物技术教育部重点实验室无锡 214122;
2. 江南大学药学院无锡 214122;
3. 江苏省微生物研究所有限公司无锡 214063

Cloning, Expressing of the prpC2 Gene Encoding Citrate Synthase from Corynebacterium crenatum and Its Effect on L-arginine Synthesis

FANG Zhan¹, XU Mei-juan¹, RAO Zhi-ming¹, MAN Zai-wei¹, XU Zheng-hong², GENG Yan², LU Mao-lin³

1. The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China;
2. School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China;
3. Jiangsu Institute of Microbiology Corporation Limited, Wuxi 214063, China

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摘要：

钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5是诱变选育的一株高产精氨酸菌株.柠檬酸合酶作为TCA途径的关键酶,对细胞胞内氨基酸代谢流调节有重要作用.在钝齿棒杆菌SYPA5-5中过量表达同源的柠檬酸合酶(citrate synthase)基因 prpC2 ,研究其对精氨酸及副产物合成的影响.重组菌C. crenatum SYPA5-5/pDXW-10-prpC2 胞内柠檬酸合酶比酶活提高了5.37倍,使L-精氨酸产量在5L发酵罐中达到44.7g/L,与对照相比提高了23.1%.同时,有机酸测定分析TCA循环的精氨酸前体柠檬酸及异柠檬酸的量有所提高,且赖氨酸合成前体草酰乙酸量减少,氨基酸测定分析L-精氨酸发酵中最主要的副产物L-赖氨酸浓度由原来的5.96g/L降到1.21g/L,降低了80%.

关键词： L-精氨酸; 钝齿棒杆菌; 柠檬酸合酶; 发酵

Abstract:

Corynebacterium crenatum SYPA5-5 is an L-arginine high-producing industrial strain of mutation breeding. The role of citrate synthase in L-arginine biosynthesis was investigated by overexpressing the citrate synthase (prpC2) gene in C. crenatum SYPA5-5. The resultant 5.37-fold increase in intracellular citrate synthase activity was achieved in the prpC2-overexpressing strain C. crenatum SYPA5-5/pDXW-10-prpC2 . The recombinant strain enhanced the L-arginine yield to 44.7g/L by about 23.1% in 5L fermenter, as compared to the control, with affecting glucose depletion rate slightly. While the L-arginine yield increased in the prpC2-overexpressing strain, the L-lysine yield, the most primary by-product formation during L-arginine fermentation, decreased to 1.21g/L from the original concentration 5.96g/L, correlating with an increase in the tricarboxylic acid cycle (TCA) intermediates (citrate and isocitrate) and an increase in the activity of citrate synthase.

Key words: L-arginine Corynebacterium crenatum Citrate synthase Fermentation

收稿日期: 2014-12-31 出版日期: 2015-03-25

ZTFLH:

Q78

基金资助:

国家"973"计划(2012CB725202),国家"863"计划(2012AA022102),国家自然科学青年基金(31300028),教育部重点研究项目(113033A),江苏省青年自然科学基金(BK20130137),中央高校基本科研业务费专项资金(JUSRP51306A)资助项目

通讯作者: 饶志明;陆茂林 E-mail: raozhm@jiangnan.edu.cn;lumaolin@jsim.cn

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引用本文:

房战, 徐美娟, 饶志明, 满在伟, 许正宏, 耿燕, 陆茂林. 过量表达钝齿棒杆菌柠檬酸合酶编码基因prpC2对L-精氨酸合成的影响[J]. 中国生物工程杂志, 2015, 35(3): 49-55.

FANG Zhan, XU Mei-juan, RAO Zhi-ming, MAN Zai-wei, XU Zheng-hong, GENG Yan, LU Mao-lin. Cloning, Expressing of the prpC2 Gene Encoding Citrate Synthase from Corynebacterium crenatum and Its Effect on L-arginine Synthesis. China Biotechnology, 2015, 35(3): 49-55.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150307 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I3/49

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