caa a22 4500 605505799 CHVBK 20210128100623.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6675-1 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6675-1 Highly efficient enzymatic synthesis of tert -butyl ( S )-6-chloro-5-hydroxy-3-oxohexanoate with a mutant alcohol dehydrogenase of Lactobacillus kefir [Elektronische Daten] [Xiu-Juan He, Shao-Yun Chen, Jian-Ping Wu, Li-Rong Yang, Gang Xu] 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 (1.27U/mg) over LbADH (0.34U/mg) and wild-type LkADH (0.03U/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 427mM (100g/L) gave (S)-CHOH in 94% yield and 99.5% e.e. after a reaction time of 38h with whole cells expressing LkTADH. The space-time yield and turnover number of NADP+ in this process were 10.6mmol/L/h and 16,060mol/mol, respectively, which were the highest values ever reported. This new approach therefore represents a promising alternative for the efficient synthesis of (S)-CHOH. Springer-Verlag Berlin Heidelberg, 2015 Lactobacillus kefir alcohol dehydrogenase nationallicence tert -Butyl ( S )-6-chloro-5-hydroxy-3-oxohexanoate nationallicence Statins nationallicence Engineered enzyme nationallicence Fed-batch strategy nationallicence He Xiu-Juan Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, 310027, Hangzhou, People's Republic of China aut Chen Shao-Yun College of Life Science, Zhejiang Chinese Medical University, 548# Binwen Road, 310053, Hangzhou, People's Republic of China aut Wu Jian-Ping Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, 310027, Hangzhou, People's Republic of China aut Yang Li-Rong Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, 310027, Hangzhou, People's Republic of China aut Xu Gang Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, 310027, Hangzhou, People's Republic of China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 8963-8975 0175-7598 99:21<8963 2015 99 253 https://doi.org/10.1007/s00253-015-6675-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6675-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- He Xiu-Juan Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, 310027, Hangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Chen Shao-Yun College of Life Science, Zhejiang Chinese Medical University, 548# Binwen Road, 310053, Hangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Wu Jian-Ping Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, 310027, Hangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Yang Li-Rong Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, 310027, Hangzhou, People's Republic of China aut NATIONALLICENCE 700 1- Xu Gang Institute of Bioengineering, College of Chemical and Biochemical Engineering, Zhejiang University, 38# Zheda Road, 310027, Hangzhou, People's Republic of China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 8963-8975 0175-7598 99:21<8963 2015 99 253