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

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

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. 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 Jianzhong^1,2, MAO Zongwan²

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/Abstract

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 P_T5 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 P_T5 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

黄明涛, 王玥, 刘建忠, 毛宗万. Multiple Strategies for Metabolic Engineering of Escherichia coli for Efficient Production of Coenzyme Q₁₀[J]. , 2011, 19(2): 316-326.

HUANG Mingtao, WANG Yue, LIU Jianzhong, MAO Zongwan. Multiple Strategies for Metabolic Engineering of Escherichia coli for Efficient Production of Coenzyme Q₁₀[J]. , 2011, 19(2): 316-326.

参考文献

1 Cluis, C.P., Burja, A.M., Martin, V.J.J., “Current prospects for the production of coenzyme Q₁₀ in microbes”, Trends Biotechnol., 25, 514-521 (2007).
2 Jeya, M., Moon, H.J., Lee, J.L., Kim, I.W., Lee, J.K., “Current state of coenzyme Q₁₀ production and its applications”, Appl. Microbiol. Biotechnol., 85, 1653-1663 (2010).
3 Meganathan, R., “Ubiquinone biosynthesis in microorganisms”, FEMS Microbiol. Lett., 203, 131-139 (2001).
4 Okada, K., Suzuki, K., Kamiya, Y., Zhu, X., Fujisaki, S., Nishimura, Y., Nishino, T., Nakagawad, T., Kawamukai, M., Matsuda, H., “Polyprenyl diphosphate synthase essentially defines the length of the side chain of ubiquinone”, Biochim. Biophys. Acta, 1302, 217-223 (1996).
5 Okada, K., Kamiya, Y., Zhu, X., Suzuki, K., Tanaka, K., Nakagawa, T., Matsuda, H., Kawamukai, M., “Cloning of the sdsA gene encoding solanesyl diphosphate synthase from Rhodobacter capsulatus and its functional expression in Escherichia coli and Saccharomyces cerevisiae”, J. Bacteriol., 179, 5992-5998 (1997).
6 Takahashi, S., Ogiyama, Y., Kusano, H., Shimada, H., Kawamukai, M., Kadowaki, K., “Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant”, FEBS Lett., 580, 955-959 (2006).
7 Barkovich, R., Liao, J.C., “Metabolic engineering of isoprenoids”, Metab. Eng., 3, 27-39 (2001).
8 Lee, J.K., Her, G., Kim, S.Y., Seo, J.H., “Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens”, Biotechnol. Prog., 20, 51-56 (2004).
9 Okada, K., Kainou, T., Tanaka, K., Nakagawa, T., Matsuda, H., Kawamukai, M., “Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans”, Eur. J. Biochem., 255, 52-59 (1998).
10 Frei, B., Kim, M.C., Ames, B.N., “Ubiquinol-10 is an effective lipid-soluble antioxidant at physiological concentrations”, Proc. Natl. Acad. Sci. USA, 87, 4879-4883 (1990).
11 Kawamukai, M., “Biosynthesis, bioproduction and novel roles of ubiquinone”, J. Biosci. Bioeng., 94, 511-517 (2002).
12 Langsjoen, P.H., Langsjoen, A.M., “Coenzyme Q₁₀ in cardiovascular disease with emphasis on heart failure and myocardial ischaemia”, Asia Pacific Heart J., 7, 160-168 (1998).
13 Negishi, E., Liou, S.Y., Xu, C.D., Huo, S.Q., “A novel, highly selective, and general methodology for the synthesis of 1,5-diene-containing oligoisoprenoids of all possible geometrical combinations exemplified by an iterative and convergent synthesis of coenzyme Q₁₀ ”, Org. Lett., 4, 261-264 (2002).
14 Lipshutz, B.H., Mollard, P., Pfeiffer, S.S., Chrisman, W., “A short, highly efficient synthesis of coenzyme Q₁₀ ”, J. Am. Chem. Soc., 124, 14282-14823 (2002).
15 Park, Y.C., Kim, S.J., Choi, J.H., Lee, W.H., Park, K.M., Kawamukai, M., Ryu, Y.W., Seo, J.H., “Batch and fed-batch production of coenzyme Q₁₀ in recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Gluconobacter suboxydans”, Appl. Microbiol. Biotechnol., 67, 192-196 (2005).
16 Yoshida, H., Kotani, Y., Ochiai, K., Araki, K., “Production of ubiquinone-10 using bacteria”, J. Gen. Appl. Microbiol., 44, 19-26 (1998).
17 Ha, S.J., Kim, S.Y., Seo, J.H., Jeya, M., Zhang, Y.W., Ramu, T., Kim, I.W., Lee, J.K., “Ca²⁺ increases the specific coenzyme Q₁₀ content in Agrobacterium tumefaciens”, Bioprocess Biosyst. Eng., 32, 697-700 (2010).
18 Sakato, K., Tanaka, H., Shibata, S., Kuratsu, Y., “Agitation-aeration studies on coenzyme Q₁₀ production using Rhodopseudomonas spheroids”, Biotechnol. Appl. Biochem., 16, 19-22 (1992).
19 Jiang, S.Y., Yu, L.J., Shen, X.L., Xiong, X., Jian, Y., “Effects of aromatic amino acids on the biosynthesis of CoQ₁₀ in Rhodopseudomonas palustris”, Pharmaceut. Biotechnol., 15 (5), 380-387 (2008).
20 Zahiri, H.S., Yoon, S.H., Keasling, J.D., Lee, S.H., Kim, S.W., Yoon, S.C., Shin, Y.C., “Coenzyme Q₁₀ production in recombinant Escherichia coli strains engineered with a heterologous decaprenyl diphosphate synthase gene and foreign mevalonate pathway”, Metab. Eng., 8, 406-416 (2006).
21 Takahashi, S., Nishino, T., Koyam, T., “Isolation and expression of Paracoccus denitrificans decaprenyl diphosphate synthase gene for production of ubiquinone-10 in Escherichia coli”, Biochem. Eng. J., 16,183-190 (2003).
22 Zahiri, H.S., Noghabi, K.A., Shin, Y.C., “Biochemical characterization of the decaprenyl diphosphate synthase of Rhodobacter sphaeroides for coenzyme Q₁₀ production”, Appl. Microbiol. Biotechnol., 73, 796-806 (2006).
23 Harker, M., Bramley, P.M., “Expression of prokaryotic 1-deoxy-Dxyluose-5-phosphatase in Escherichia coli increases carotenoid and ubiquinone biosynthesis”, FEBS Lett., 87, 115-119 (1999).
24 Kim, S.J., Kim, M.D., Choi, J.H., Kim, S.Y., Ryu, Y.W., Seo, J.H., “Amplification of 1-deoxy-D-xyluose 5-phosphate (DXP) synthase level increases coenzyme Q₁₀ production in recombinant Escherichia coli”, Appl. Microbiol. Biotechnol., 72, 982-985 (2006).
25 Choi, J.H., Ryu, Y.W., Park, Y.C., Seo, J.H., “Synergistic effects of chromosomal ispB deletion and dxs overexpression on coenzyme Q₁₀ production in recombinant Escherichia coli expressing Agrobacterium tumefaciens dps gene”, J. Biotechnol., 144, 64-69 (2009).
26 Kwon, O., Druce-Hoffman, M., Meganathan, R., “Regulation of the ubiquinone (coenzyme Q) biosynthetic genes ubiCA in Escherichia coli”, Curr. Microbiol., 50, 180-189 (2005).
27 Zhang, D., Li, Z., Wang, F., Shrestha, B., Tian, P., Tan, T., “Expression of various genes to enhance ubiquinone metabolic pathway in Agrobacterium tumefaciens”, Enzyme Microb. Technol., 41, 772-779 (2007).
28 Zhu, X., Yuasa, M., Okada, K., Suzuki, K., Nakagawa, T., Kawamukai, M., Matsuda, H., “Production of ubiquinone in Escherichia coli by expression of various genes responsible for ubiquinone biosynthesis”, J. Ferment. Bioeng., 79, 493-495 (1995).
29 Ye, J., Ma, L., Wu, H., Liu, X., Zhang, H., “Effect of overexpressing ubiCA genes responsible for ubiquinone biosynthesis on ubiquinone production in Escherichia coli”, J. East China Univ. Sci. Technol. (Natural Science Edition), 32, 269-273 (2006).
30 Farmer, W.R., Liao, J.C., “Precursor balancing for metabolic engineering of lycopene production in Escherichia coli”, Biotechnol. Prog., 17, 57-61 (2001).
31 Martínez, A., Zhu, J., Lin, H., Bennett, G.N., San, K.Y., “Replacing Escherichia coli NAD-dependent glyceraldehydes 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways”, Metab. Eng., 10, 352-359 (2008).
32 Jones, K.L., Kim, S.W., Keasling, J.D., “Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria”, Metab. Eng., 2, 328-338 (2000).
33 Guzman, L.M., Belin, D., Carson, M.J., Beckwith, J., “Tight regulation, modulation, and high-level expression by vectors containing the arabinose P BAD promoter”, J. Bacteriol., 177, 4121-4130 (1995).
34 Datsenko, K.A., Wanner, B.L., “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products”, Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000).
35 Kedar, P., Colah, R., Shimizu, K., “Proteomic investigation on the pyk-F gene knockout Escherichia coli for aromatic amino acid production”, Enzyme Microb. Technol., 41, 455-465 (2007).
36 Lee, J.K., Oh, D.K., Kim, S.Y., “Cloning and characterization of the dxs gene, encoding 1-deoxy-d-xylulose 5-phosphate synthase from Agrobacterium tumefaciens, and its overexpression in Agrobacterium tumefaciens”, J. Biotechnol., 128, 555-566 (2007).
37 Lee, P.C., Mijts, B.N., Schmidt-Dannert, C., “Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered Escherichia coli”, Appl. Microbiol. Biotechnol., 65, 538-546 (2004).
38 Tatarko, M., Romeo, T., “Disruption of global regulatory gene to enhance central carbon flux into phenylalanine biosynthesis in Escherichia coli”, Curr. Microbiol., 43, 26-32 (2001).
39 Yakandawala, N., Romeo, T., Friesen, A.D., Madhyastha, S., “Metabolic engineering of Escherichia coli to enhance phenylalanine production”, Appl. Microbiol. Biotechnol., 78, 283-291 (2008).
40 Weilbacher, T., Suzuki, K., Dubey, A.K., Wang, X., Gudapaty, S., Morozov, I., Baker, C.S., Georgellis, D., Babitzke, P., Romeo, T., “A novel sRNA component of the carbon storage regulatory system of Escherichia coli”, Mol. Microbiol., 48, 657-670 (2003).
41 Kim, S.W., Keasling, J.D., “Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production”, Biotechnol. Bioeng., 72, 408-415 (2001).
42 O'Connor, M., Peifer, M., Bender, W., “Construction of large DNA segments in Escherichia coli”, Science, 244, 1307-1312 (1989).
43 Noack, D., Roth, M., Geuther, R., Muller, G., Undisz, K., Hoffmeier, C., Gaspar, S., “Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 strains grown in a chemostat”, Mol. Gen. Genet., 184, 121-124 (1981).
44 Birnbaum, S., Bailey, J.E., “Plasmid presence changes the relative levels of many host cell proteins and ribosome components in recombinant Escherichia coli”, Biotechnol. Bioeng., 37, 736-745 (1991).
45 Vind, J., Sorensen, M.A., Rasmussen, M.D., Pedersen, S., “Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes”, J. Mol. Biol., 231, 678-688 (1993).
46 Shiloach, J., Fass, R., “Growing E. coli to high cell density-A historical perspective on method development”, Biotechnol. Adv., 23, 345-357 (2005).

[1]	Cui Yang, Xianzhong Chen, Junzhuang Chang, Lihua Zhang, Wei Xu, Wei Shen, You Fan. Reconstruction of tyrosol synthetic pathways in Escherichia coli[J]. Chinese Journal of Chemical Engineering, 2018, 26(12): 2615-2621.
[2]	Zhibo Xu, Zhenquan Lin, Zhiwen Wang, Tao Chen. Improvement of the riboflavin production by engineering the precursor biosynthesis pathways in Escherichia coli[J]. Chinese Journal of Chemical Engineering, 2015, 23(11): 1834-1839.
[3]	Cuiwei Wang, Zhe Li, Aamir Rasool, Hongnan Qu, Dazhang Dai, Chun Li. Characterization of promoters in Escherichia coli and application for xylitol synthesis[J]. Chinese Journal of Chemical Engineering, 2015, 23(1): 234-240.
[4]	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.
[5]	郭佳庆, 骆艳娥, 范代娣, 高鹏飞, 马晓轩, 朱晨辉. Analysis of Metabolic Products by Response Surface Methodology for Production of Human-like Collagen II[J]. , 2010, 18(5): 830-836.
[6]	高鹏飞, 范代娣, 骆艳娥, 马晓轩, 马沛, 惠俊峰, 朱晨辉. Efficient and Comprehensive Utilization of Hemicellulose in the Corn Stover[J]. , 2009, 17(2): 350-354.
[7]	韩云, 李志敏, 杜鹏, 甘人宝, 叶勤. 补料分批培养耐乙酸大肠杆菌生产人表皮生长因子[J]. , 2007, 15(5): 760-764.
[8]	卢英华, 邓旭, 程志敬, 李清彪, 刘刚. 运用试验设计方法提高胞外杂合b-葡聚糖酶在重组大肠杆菌中的表达[J]. , 2007, 15(2): 172-177.
[9]	花秀夫, 范代娣, 骆艳娥, 张兮, 施惠娟, 米钰, 马晓轩, 尚龙安, 赵桂仿. 重组大肠杆菌分批-补料高密度发酵生产类人胶原蛋白的动力学 [J]. , 2006, 14(2): 242-247.

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

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

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 9

编辑推荐

Metrics

本文评价