SCI和EI收录∣中国化工学会会刊

›› 2011, Vol. 19 ›› Issue (2): 316-326.

• • 上一篇    下一篇

Multiple Strategies for Metabolic Engineering of Escherichia coli for Efficient Production of Coenzyme Q10

黄明涛1, 王玥1, 刘建忠1,2, 毛宗万2   

  1. 1. The Key Laboratory of Gene Engineering of Ministry of Education, State Key Laboratory of Biocontrol, Sun Yat-Sen University, Guangzhou 510275, China;
    2. MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
  • 收稿日期:2010-09-28 修回日期:2011-01-30 出版日期:2011-04-28 发布日期:2011-04-28
  • 通讯作者: LIU Jianzhong,MAO Zongwan,E-mail:lssljz@mail.sysu.edu.cn
  • 基金资助:
    Supported by the National Natural Science Foundation of China(30970089,200876181,20831006);the Natural Science Foundation of Guangdong Province(9351027501000003);the Project of Science and Technology of Guangdong Province(2007A010900001)

Multiple Strategies for Metabolic Engineering of Escherichia coli for Efficient Production of Coenzyme Q10

HUANG Mingtao1, WANG Yue1, LIU Jianzhong1,2, MAO Zongwan2   

  1. 1. The Key Laboratory of Gene Engineering of Ministry of Education, State Key Laboratory of Biocontrol, Sun Yat-Sen University, Guangzhou 510275, China;
    2. MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, Sun Yat-Sen University, Guangzhou 510275, China
  • Received:2010-09-28 Revised:2011-01-30 Online:2011-04-28 Published:2011-04-28
  • Supported by:
    Supported by the National Natural Science Foundation of China(30970089,200876181,20831006);the Natural Science Foundation of Guangdong Province(9351027501000003);the Project of Science and Technology of Guangdong Province(2007A010900001)

摘要: Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli (△ispB::ddsA,△pykFA and △gapA::gapC),harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.

关键词: coenzyme Q10, Escherichia coli, gene replacement, NADPH availability, precursor balance

Abstract: Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli (△ispB::ddsA,△pykFA and △gapA::gapC),harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.

Key words: coenzyme Q10, Escherichia coli, gene replacement, NADPH availability, precursor balance