title: Determination of key enzymes for threonine synthesis through in vitro metabolic pathway analysis.
abstract: BACKGROUND
  The overexpression of key enzymes in a metabolic pathway is a frequently used genetic engineering strategy for strain improvement. Metabolic control analysis has been proposed to quantitatively determine key enzymes. However, the lack of quality data often makes it difficult to correctly identify key enzymes through control analysis. Here, we proposed a method combining in vitro metabolic pathway analysis and proteomics measurement to find the key enzymes in threonine synthesis pathway.
  
  
  RESULTS
  All enzymes in the threonine synthesis pathway were purified for the reconstruction and perturbation of the in vitro pathway. Label-free proteomics technology combined with APEX (absolute protein expression measurements) data analysis method were employed to determine the absolute enzyme concentrations in the crude enzyme extract obtained from a threonine production strain during the fastest threonine production period. The flux control coefficient of each enzyme in the pathway was then calculated by measuring the flux changes after titration of the corresponding enzyme. The isoenzyme LysC catalyzing the first step in the pathway has the largest flux control coefficient, and thus its concentration change has the biggest impact on pathway flux. To verify that the key enzyme identified through in vitro pathway analysis is also the key enzyme in vivo, we overexpressed LysC in the original threonine production strain. Fermentation results showed that the threonine concentration was increased 30% and the yield was increased 20%.
  
  
  CONCLUSIONS
  In vitro metabolic pathways simulating in vivo cells can be built based on precise measurement of enzyme concentrations through proteomics technology and used for the determination of key enzymes through metabolic control analysis. This provides a new way to find gene overexpression targets for industrial strain improvement.
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. zhang_yf1@tib.cas.cn.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. mql198868@tju.edu.cn.|Key Laboratory of Systems Bioengineering, Ministry of Education, Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China. mql198868@tju.edu.cn.
authors.forename: Qinglong
authors.initials: Q
authors.lastname: Meng
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. ma_hw@tib.cas.cn.
authors.forename: Hongwu
authors.initials: H
authors.lastname: Ma
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. liu_yf@tib.cas.cn.
authors.forename: Yongfei
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. cao_gq@tib.cas.cn.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Cao
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. zhang_xr@tib.cas.cn.
authors.forename: Xiaoran
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. zheng_p@tib.cas.cn.
authors.forename: Ping
authors.initials: P
authors.lastname: Zheng
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. sun_jb@tib.cas.cn.
authors.forename: Jibin
authors.initials: J
authors.lastname: Sun
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. zhang_dw@tib.cas.cn.
authors.forename: Dawei
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. jiang_wx@tib.cas.cn.
authors.forename: Wenxia
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People's Republic of China. ma_yh@tib.cas.cn.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D053898:Biosynthetic Pathways; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D013912:Threonine / Q000096:biosynthesis*
chemical_list: D029968:Escherichia coli Proteins; D013912:Threonine