title: In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology.
abstract: In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model's predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals.
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Fang
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Huina
authors.initials: H
authors.lastname: Dong
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Cai
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zheng
authors.affiliation: Sino-German Joint Research Institute, Nanchang University, Nanchang, Jiangxi, China.
authors.forename: Haixing
authors.initials: H
authors.lastname: Li
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.|Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Jibin
authors.initials: J
authors.lastname: Sun
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014578:Uroporphyrins / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D004798:Enzymes; D011994:Recombinant Proteins; D014578:Uroporphyrins; C063209:15,23-dihydrosirohydrochlorin