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[1]董晓翔,陈修来,刘立明,等.代谢工程改造Escherichia coli生产L-苹果酸[J].应用与环境生物学报,2017,23(02):269-275.[doi:10.3724/SP.J.1145.2016.04012]
 DONG Xiaoxiang,,et al.Design and construction of an L-malate-overproducing Escherichia coli strain[J].Chinese Journal of Applied & Environmental Biology,2017,23(02):269-275.[doi:10.3724/SP.J.1145.2016.04012]
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代谢工程改造Escherichia coli生产L-苹果酸()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
23卷
期数:
2017年02期
页码:
269-275
栏目:
研究论文
出版日期:
2017-04-25

文章信息/Info

Title:
Design and construction of an L-malate-overproducing Escherichia coli strain
作者:
董晓翔陈修来刘立明史仲平
1江南大学食品科学与技术国家重点实验室 无锡 214122 2江南大学工业生物技术教育部重点实验室 无锡 214122 3江南大学食品微生物制造工程实验室 无锡 214122
Author(s):
DONG Xiaoxiang1 2 3 CHEN Xiulai1 2 3 LIU Liming1 2 3 & SHI Zhongping2*
1State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China 2Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China 3Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi 214122, China
关键词:
大肠杆菌L-苹果酸苹果酸酶NADH激酶代谢工程基因敲除
Keywords:
Escherichia coli L-malate malic enzyme NADH kinase metabolic engineering gene knockout
分类号:
Q78 : TQ921
DOI:
10.3724/SP.J.1145.2016.04012
摘要:
为了高效生产L-苹果酸,首先在大肠杆菌w3110中敲除ldhA、poxB、pflB和pta-ackA基因积累丙酮酸,为L-苹果酸合成提供前体,并且通过苹果酸酶的引入构建L-苹果酸一步合成路径,将丙酮酸转化为L-苹果酸. 在此基础上,敲除frdBC、fumB和fumAC阻断L-苹果酸代谢路径,并结合pos5基因的表达对胞内辅因子路径进行优化. 结果表明:(1)ldhA、poxB、pflB和pta-ackA基因的敲除能有效地提高丙酮酸产量到20.9 g/L;(2)苹果酸酶突变及过量表达使得L-苹果酸和琥珀酸产量分别提高了87.2%和31.6%,达到1.46 g/L和3.25 g/L;(3)通过敲除frdBC、fumB和fumAC,L-苹果酸产量增加到3.42 g/L;(4)pos5基因的表达降低了胞内NADH/NAD+比率,增加了NADPH含量,最终突变菌株Escherichia coli F0921的L-苹果酸产量达到9.34 g/L. 因此,通过苹果酸酶构建L-苹果酸生物合成路径提高L-苹果酸的生产是可行的,结果可为代谢工程改造大肠杆菌生产L-苹果酸提供了新的研究思路. (图4 表4 参21)
Abstract:
The aim of our study was to engineer Escherichia coli to produce L-malate. The pool of pyruvate was first increased by knocking out the genes ldhA, poxB, pflB, and pta-ackA in E. coli w3110, and the one-step L-malate synthesis pathway was constructed via malic enzyme. Furthermore, the L-malate consumption pathway was disrupted by knocking out frdBC, fumB, and fumAC, and the cofactor level was optimized by overexpressing pos5. The pyruvate production was increased to 20.9 g/L with knockout of ldhA, poxB, pflB, and pta-ackA, and the titers of L-malate and succinate were respectively increased to 87.2% and 31.6% with overexpression of C490S. The titer of l-malate was further improved to 9.34 g/L in E. coli F0921 with knockout of frdBC, fumB, and fumAC, and optimization of the cofactor level. In summary, the one-step L-malate synthesis pathway was successfully constructed in E. coli to produce L-malate by malic enzyme, providing a new method to produce L-malate.

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更新日期/Last Update: 2017-04-25