title: Highly efficient enzymatic synthesis of tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate with a mutant alcohol dehydrogenase of Lactobacillus kefir.
abstract: tert-Butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) is a valuable chiral synthon, which is used for the synthesis of the cholesterol-lowering drugs atorvastatin and rosuvastatin. To date, only the alcohol dehydrogenases from Lactobacillus brevis (LbADH) and Lactobacillus kefir (LkADH) have demonstrated catalytic activity toward the asymmetric reduction of tert-butyl 6-chloro-3,5-dioxohexanoate (CDOH) to (S)-CHOH. Herein, a tetrad mutant of LkADH (LkTADH), A94T/F147L/L199H/A202L, was screened to be more efficient in this bioreduction process, exhibiting a 3.7- and 42-fold improvement in specific activity toward CDOH (1.27 U/mg) over LbADH (0.34 U/mg) and wild-type LkADH (0.03 U/mg), respectively. The molecular basis for the improved catalytic activity of LkTADH toward CDOH was investigated using homology modeling and docking analysis. Two major issues had a significant impact on the biocatalytic efficiency of this process, including (i) the poor aqueous stability of the substrate and (ii) partial substrate inhibition. A fed-batch strategy was successfully developed to address these issues and maintain a suitably low substrate concentration throughout the entire process. Several other parameters were also optimized, including the pH, temperature, NADP(+) concentration and cell loading. A final CDOH concentration of 427 mM (100 g/L) gave (S)-CHOH in 94 % yield and 99.5 % e.e. after a reaction time of 38 h with whole cells expressing LkTADH. The space-time yield and turnover number of NADP(+) in this process were 10.6 mmol/L/h and 16,060 mol/mol, respectively, which were the highest values ever reported. This new approach therefore represents a promising alternative for the efficient synthesis of (S)-CHOH.
authors.affiliation: Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, Hangzhou, 310027, People's Republic of China.
authors.forename: Xiu-Juan
authors.initials: XJ
authors.lastname: He
authors.affiliation: College of Life Science, Zhejiang Chinese Medical University, 548# Binwen Road, Hangzhou, 310053, People's Republic of China.
authors.forename: Shao-Yun
authors.initials: SY
authors.lastname: Chen
authors.affiliation: Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, Hangzhou, 310027, People's Republic of China. wjp@zju.edu.cn.
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Wu
authors.affiliation: Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, Hangzhou, 310027, People's Republic of China. lryang@zju.edu.cn.
authors.forename: Li-Rong
authors.initials: LR
authors.lastname: Yang
authors.affiliation: Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, Hangzhou, 310027, People's Republic of China.
authors.forename: Gang
authors.initials: G
authors.lastname: Xu
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D002208:Caproates / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D011487:Protein Conformation
chemical_list: D002208:Caproates; D050505:Mutant Proteins; D000426:Alcohol Dehydrogenase
keywords: Engineered enzyme; Fed-batch strategy; Lactobacillus kefir alcohol dehydrogenase; Statins; tert-Butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate