title: Improving the Production of L-Phenylalanine by Identifying Key Enzymes Through Multi-Enzyme Reaction System in Vitro.
abstract: L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product.
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.|Department of Food Science and Engineering, School of Food, Nanchang University, Nanchang 330029, People's Republic of China.
authors: Dongqin
authors: D
authors: Ding
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.
authors: Yongfei
authors: Y
authors: Liu
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.
authors: Yiran
authors: Y
authors: Xu
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.
authors: Ping
authors: P
authors: Zheng
authors: Department of Food Science and Engineering, School of Food, Nanchang University, Nanchang 330029, People's Republic of China.
authors: Haixing
authors: H
authors: Li
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.
authors: Dawei
authors: D
authors: Zhang
authors: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, People's Republic of China.
authors: Jibin
authors: J
authors: Sun
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000378:metabolism; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D010649:Phenylalanine / Q000096:biosynthesis* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012765:Shikimic Acid / Q000378:metabolism
chemical_list: D004798:Enzymes; D012765:Shikimic Acid; D010649:Phenylalanine; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase