Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q10

doi:10.1016/S1004-9541(14)60082-3

Chinese Journal of Chemical Engineering ›› 2014, Vol. 22 ›› Issue (5): 559-569.DOI: 10.1016/S1004-9541(14)60082-3

• 催化、动力学与反应工程 • 上一篇下一篇

Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q₁₀

黄明涛, 陈韵妍, 刘建忠

Biotechnology Research Center and MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Life Science, Sun Yat-Sen University, Guangzhou 510275, China

收稿日期:2012-12-10 修回日期:2013-05-10 出版日期:2014-05-28 发布日期:2014-05-06
通讯作者: LIU Jianzhong,E-mail: lssljz@mail.sysu.edu.cn
基金资助:
Supported by the National Natural Science Foundation of China (30970089, 20876181, 21276289) and the Natural Science Foundation of Guangdong Province (9351027501000003, S2011010001396).

Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q₁₀

HUANG Mingtao, CHEN Yunyan, LIU Jianzhong

Biotechnology Research Center and MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Life Science, Sun Yat-Sen University, Guangzhou 510275, China

Received:2012-12-10 Revised:2013-05-10 Online:2014-05-28 Published:2014-05-06
Supported by:
Supported by the National Natural Science Foundation of China (30970089, 20876181, 21276289) and the Natural Science Foundation of Guangdong Province (9351027501000003, S2011010001396).

摘要/Abstract

摘要： The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E. coli strain for coenzyme Q₁₀ production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution (multiple copy integration) and replicon-free and markerless chromosomal integration (single copy integration), respectively. A coenzyme Q₁₀ hyper-producer Escherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution, replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway. The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass (DCM) of coenzyme Q₁₀ when supplemented with 0.075 g·L^-1 of 4-hydroxy benzoic acid; this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q₁₀ production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.

关键词: coenzyme Q₁₀, Escherichia coli, chemically inducible chromosomal evolution, replicon-free and markerless chromosomal integration, chromosomal engineering

Abstract: The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E. coli strain for coenzyme Q₁₀ production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution (multiple copy integration) and replicon-free and markerless chromosomal integration (single copy integration), respectively. A coenzyme Q₁₀ hyper-producer Escherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution, replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway. The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass (DCM) of coenzyme Q₁₀ when supplemented with 0.075 g·L^-1 of 4-hydroxy benzoic acid; this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q₁₀ production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.

Key words: coenzyme Q₁₀, Escherichia coli, chemically inducible chromosomal evolution, replicon-free and markerless chromosomal integration, chromosomal engineering

黄明涛, 陈韵妍, 刘建忠. Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q₁₀[J]. Chinese Journal of Chemical Engineering, 2014, 22(5): 559-569.

HUANG Mingtao, CHEN Yunyan, LIU Jianzhong. Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q₁₀[J]. Chin.J.Chem.Eng., 2014, 22(5): 559-569.

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Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q₁₀

Chromosomal Engineering of Escherichia coli for Efficient Production of Coenzyme Q₁₀

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