title: Production of 5-aminolevulinic acid by cell free multi-enzyme catalysis.
abstract: 5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160min, ALA concentration was increased to 5.4mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production.
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, PR China; Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China.
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, PR China.
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, PR China.
authors.forename: Xiaozhi
authors.initials: X
authors.lastname: Ju
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, PR China.
authors.forename: Chunling
authors.initials: C
authors.lastname: Ma
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, PR China. Electronic address: 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, PR China.
authors.forename: Jiuzhou
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, PR China.
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, PR China.
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, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, PR China.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China.
authors.forename: Xueming
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, PR China. Electronic address: chentao@tju.edu.cn.
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
mesh_terms: D000622:Aminolevulinic Acid / Q000378:metabolism*; D061249:Batch Cell Culture Techniques; D055162:Biocatalysis; D001709:Biotechnology / Q000379:methods*; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000378:metabolism*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism; D013379:Substrate Specificity; D019802:Succinic Acid / Q000378:metabolism
chemical_list: D004798:Enzymes; D000622:Aminolevulinic Acid; D019802:Succinic Acid; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase
keywords: 5-Aminolevulinic acid; 5-Aminolevulinic acid synthase; ATP regeneration; Cell free production; Fed-batch