title: Fumarate Production by Torulopsis glabrata: Engineering Heterologous Fumarase Expression and Improving Acid Tolerance.
abstract: Fumarate is a well-known biomass building block compound. However, the poor catalytic efficiency of fumarase is one of the major factors preventing its widespread production. To address this issue, we selected residues 159HPND162 of fumarase from Rhizopus oryzae as targets for site-directed mutagenesis based on molecular docking analysis. Twelve mutants were generated and characterized in detail. Kinetic studies showed that the Km values of the P160A, P160T, P160H, N161E, and D162W mutants were decreased, whereas Km values of H159Y, H159V, H159S, N161R, N161F, D162K, and D162M mutants were increased. In addition, all mutants displayed decreased catalytic efficiency except for the P160A mutant, whose kcat/Km was increased by 33.2%. Moreover, by overexpressing the P160A mutant, the engineered strain T.G-PMS-P160A was able to produce 5.2 g/L fumarate. To further enhance fumarate production, the acid tolerance of T.G-PMS-P160A was improved by deleting ade12, a component of the purine nucleotide cycle, and the resulting strain T.G(△ade12)-PMS-P160A produced 9.2 g/L fumarate. The strategy generated in this study opens up new avenues for pathway optimization and efficient production of natural products.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, China.
authors.forename: Xiulai
authors.initials: X
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Song
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, China.
authors.forename: Qiuling
authors.initials: Q
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, China.
authors.forename: Liming
authors.initials: L
authors.lastname: Liu
mesh_terms: D001665:Binding Sites; D005649:Fumarate Hydratase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D012233:Rhizopus / Q000201:enzymology / Q000235:genetics; D004718:Saccharomycetales / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature
chemical_list: D005650:Fumarates; D005649:Fumarate Hydratase