title: Efficient production of enantiomerically pure D-phenyllactate from phenylpyruvate by structure-guided design of an engineered D-lactate dehydrogenase.
abstract: 3-Phenyllactic acid (PLA) is an antimicrobial compound with broad-spectrum activity against bacteria and fungi that could be widely used in the food industry and livestock feeds. Notably, D-PLA exhibits higher antibacterial activity, which gains more attention than L-PLA. In this report, the D-lactate dehydrogenase DLDH744 from Sporolactobacillus inulinus CASD was engineered to increase the enzymatic activities toward phenylpyruvate by protein structure-guided modeling analysis. The phenylpyruvate molecule was first docked in the active center of DLDH744. The residues that might tightly pack around the benzene ring of phenylpyruvate were all selected for mutation. The single site mutant M307L showed the highest increased activity toward bulkier substrate phenylpyruvate than the wild type. By using the engineered D-lactate dehydrogenase M307L expressed in Escherichia coli strains, without coexpression of the cofactor regeneration system, 21.43 g/L D-PLA was produced from phenylpyruvate with a productivity of 1.58 g/L/h in the fed-batch biotransformation process, which ranked in the list as the highest production titer of D-PLA by D-lactate dehydrogenase. The enantiomeric excess value of produced D-PLA in the broth was higher than 99.7 %. Additionally, the structure-guided design of this enzyme will also provide referential information for further engineering other 2-hydroxyacid dehydrogenases, which are useful for a wide range of fine chemical synthesis.
authors.affiliation: School of Life Sciences, Anhui University, Hefei, 230039, People's Republic of China.|CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Lingfeng
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Hangzhou, 311121, China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Xu
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
authors.forename: Limin
authors.initials: L
authors.lastname: Wang
authors.affiliation: School of Life Sciences, Anhui University, Hefei, 230039, People's Republic of China. adamyin@163.com.
authors.forename: Ruochun
authors.initials: R
authors.lastname: Yin
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. yub@im.ac.cn.
authors.forename: Bo
authors.identifier: http://orcid.org/0000-0003-3148-0986
authors.initials: B
authors.lastname: Yu
mesh_terms: D000900:Anti-Bacterial Agents / Q000138:chemical synthesis*; D056449:Bacillales / Q000201:enzymology* / Q000235:genetics; D001711:Biotransformation; D020134:Catalytic Domain / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D045022:Lactate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D007773:Lactates / Q000138:chemical synthesis*; D009154:Mutation; D010667:Phenylpyruvic Acids / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D000900:Anti-Bacterial Agents; D007773:Lactates; D010667:Phenylpyruvic Acids; C017648:3-phenyllactic acid; D045022:Lactate Dehydrogenases; C071195:D-lactate dehydrogenase; C031606:phenylpyruvic acid
keywords: D-Lactate dehydrogenase; D-Phenyllactate; Optical purity; Protein engineering