Highly efficient enzymatic synthesis of tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate with a mutant alcohol dehydrogenase of Lactobacillus kefir

• Published in: Applied microbiology and biotechnology Vol. 99; no. 21; pp. 8963 - 8975
• Main Authors: He, Xiu-Juan, Chen, Shao-Yun, Wu, Jian-Ping, Yang, Li-Rong, Xu, Gang
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.11.2015; Springer; Springer Nature B.V
• Subjects: Alcohol; alcohol dehydrogenase; Alcohol Dehydrogenase - genetics; Alcohol Dehydrogenase - metabolism; Analysis; Anticholesteremic agents; Bacteria; biocatalysis; Bioengineering; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Caproates - metabolism; Cardiovascular disease; Catalysis; Cholesterol; Chromatography; Dehydrogenases; Enzymes; Health aspects; Influence; Lactobacillus; Lactobacillus - enzymology; Lactobacillus - genetics; Lactobacillus brevis; Lactobacillus kefiri; Life Sciences; Microbial Genetics and Genomics; Microbiology; Models, Molecular; Molecular Docking Simulation; Mutant Proteins - genetics; Mutant Proteins - metabolism; mutants; Mutation, Missense; Oxidation-Reduction; Oxidoreductases; Physiological aspects; Protein Conformation; Spacetime; Statins; Studies; temperature; China; alcohol dehydrogenase; 6-chloro-5-hydroxy-3-oxohexanoate; Fed-batch strategy; Butyl; Statins; Engineered enzyme; Lactobacillus kefir alcohol dehydrogenase; tert-Butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-6675-1

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.