title: Directed evolution and mutagenesis of glutamate decarboxylase from Lactobacillus brevis Lb85 to broaden the range of its activity toward a near-neutral pH.
abstract: Glutamate decarboxylase (GAD) transforms l-glutamate into γ-aminobutyric acid (GABA) with the consumption of a proton. GAD derived from lactic acid bacteria exhibits optimum activity at pH 4.0-5.0 and significantly loses activity at near-neutral pH. To broaden the active range of the GAD GadB1 from Lactobacillus brevis Lb85 toward a near-neutral pH, irrational design using directed evolution and rational design using site-specific mutagenesis were performed. For directed evolution of GadB1, a sensitive high-throughput screening strategy based on a pH indicator was established. One improved mutant, GadB1(T17I/D294G/Q346H), was selected from 800 variants after one round of EP-PCR. It exhibited 3.9- and 25.0-fold increase in activity and catalytic efficiency, respectively at pH 6.0. Through site-specific mutagenesis, several improved mutants were obtained, with GadB1(E312S) being the best one. The combined mutant GadB1(T17I/D294G/E312S/Q346H) showed even higher catalytic efficiency, 13.1- and 43.2-fold that of wild-type GadB1 at pH 4.6 and 6.0, respectively. The amount of GABA produced in gadB1(T17I/D294G/Q346H)-, gadB1(E312S)- and gadB1(T17I/D294G/E312S/Q346H)-expressing Corynebacterium glutamicum ATCC 13032 from endogenous l-glutamate increased by 9.6%, 20.3% and 63.9%, respectively. These results indicate that these mutations have beneficial effects on expanding the active pH range and on GABA biosynthesis, suggesting these GadB1 variants as potent candidates for GABA production.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China. Electronic address: shifeng@jiangnan.edu.cn.
authors.forename: Feng
authors.initials: F
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China.
authors.forename: Yilong
authors.initials: Y
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China.
authors.forename: Junjun
authors.initials: J
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China.
authors.forename: Nannan
authors.initials: N
authors.lastname: Wang
authors.affiliation: National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China.
authors.forename: Yongfu
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China.
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D019020:Directed Molecular Evolution; D029968:Escherichia coli Proteins / Q000235:genetics; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D052196:Levilactobacillus brevis / Q000201:enzymology* / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid; C584507:gadB protein, E coli; D005968:Glutamate Decarboxylase
keywords: Active pH range; Directed evolution; Error prone-PCR; Glutamate decarboxylase; Site-specific mutagenesis; γ-Aminobutyric acid