title: Combinatorial optimization of CO2 transport and fixation to improve succinate production by promoter engineering.
abstract: To balance the flux of an engineered metabolic pathway to achieve high yield of target product is a major challenge in metabolic engineering. In previous work, the collaborative regulation of CO2 transport and fixation was investigated with co-overexpressing exogenous genes regulating both CO2 transport (sbtA and bicA) and PEP carboxylation (phosphoenolpyruvate (PEP) carboxylase (ppc) and carboxykinase (pck)) under trc promoter in Escherichia coli for succinate biosynthesis. For balancing metabolic flux to maximize succinate titer, a combinatorial optimization strategy to fine-tuning CO2 transport and fixation process was implemented by promoter engineering in this study. Firstly, based on the energy matrix a synthetic promoter library containing 20 rationally designed promoters with strengths ranging from 0.8% to 100% compared with the widely used trc promoter was generated. Evaluations of rfp and cat reporter genes provided evidence that the synthetic promoters were stably and had certain applicability. Secondly, four designed promoters with different strengths were used for combinatorial assembly of single CO2 transport gene (sbtA or bicA) and single CO2 fixation gene (ppc or pck) expression. Three combinations, such as Tang1519 (P4 -bicA + pP19 -pck), Tang1522 (P4 -sbtA + P4 -ppc), Tang1523 (P4 -sbtA + P17 -ppc) with a more than 10% increase in succinate production were screened in bioreactor. Finally, based on the above results, co-expression of the four transport and fixation genes were further investigated. Co-expression of sbtA, bicA, and ppc with weak promoter P4 and pck with strong promoter P19 (AFP111/pT-P4 -bicA-P4 -sbtA + pACYC-P19 -pck-P4 -ppc) provided the best succinate production among all the combinations. The highest succinate production of 89.4 g/L was 37.5% higher than that obtained with empty vector control. This work significantly enhanced succinate production through combinatorial optimization of CO2 transport and fixation. The promoter engineering and combinatorial optimization strategies used herein represents a powerful approach to tailor-making metabolic pathways for the production of other industrially important chemicals. Biotechnol. Bioeng. 2016;113: 1531-1541. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan, 430068, China.|Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
authors.forename: Jun-Han
authors.initials: JH
authors.lastname: Yu
authors.affiliation: Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan, 430068, China.
authors.forename: Li-Wen
authors.initials: LW
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan, 430068, China.
authors.forename: Shi-Tao
authors.initials: ST
authors.lastname: Xia
authors.affiliation: Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan, 430068, China.
authors.forename: Hong-Mei
authors.initials: HM
authors.lastname: Li
authors.affiliation: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
authors.forename: Ya-Ling
authors.initials: YL
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
authors.forename: Xin-Hua
authors.initials: XH
authors.lastname: Liang
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.affiliation: Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan, 430068, China. yajietang@qq.com.
authors.forename: Ya-Jie
authors.initials: YJ
authors.lastname: Tang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism*; D020650:Combinatorial Chemistry Techniques; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D060847:Metabolic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013386:Succinates / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013386:Succinates; D002245:Carbon Dioxide
keywords: Escherichia coli; combinatorial optimization; metabolic balance; succinate; synthetic promoter library