title: One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.
abstract: α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; School of Food Science and Technology, Jiangnan University, Wuxi, China.
authors.forename: Gazi Sakir
authors.initials: GS
authors.lastname: Hossain
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; Synergetic Innovation Center Of Food Safety and Nutrition, Wuxi, China.
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.affiliation: School of Chemical and Biomolecular Engineeirng, Georgia Institute of Technology, Atlanta, Georgia, United States of America.
authors.forename: Hyun-dong
authors.initials: HD
authors.lastname: Shin
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; Synergetic Innovation Center Of Food Safety and Nutrition, Wuxi, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: School of Food Science and Technology, Jiangnan University, Wuxi, China.
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; Synergetic Innovation Center Of Food Safety and Nutrition, Wuxi, China.
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; Synergetic Innovation Center Of Food Safety and Nutrition, Wuxi, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D055162:Biocatalysis; D001711:Biotransformation; D003001:Cloning, Molecular; D003641:Deamination; D019020:Directed Molecular Evolution; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008190:Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008715:Methionine / Q000031:analogs & derivatives* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D021381:Protein Transport; D011514:Proteus vulgaris / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D004800:Enzymes, Immobilized; C014632:2-keto-4-methylthiobutyric acid; D008715:Methionine; D008190:Lyases