Construction Of A Carbonyl Reductases Library And Its Application For Synthesis Of Chiral Aromatic Alcohol Intermediates

Chiral aromatic alcohols such as(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol [(R)-3,5-BTPE],(S)-2-Chloro-1-(3,4-difluorophenyl)ethanol [(S)-CFPL] are crucial chiral pharmaceutical intermediates.Asymmetric reduction of chiral chiral aromatic ketones by carbonyl reductase is an important method for the synthesis of chiral aromatic alcohols.Compared with chemical methods,it has many advantages such as excellent catalytic activity and enantioselectivity.In this study,we constructed a carbonyl reductases library and use it for preparation of chiral aromatic alcohol intermediates.Firstly,we successfully constructed an enzyme library containing 17 kinds of carbonyl reductases.Primary screen sheowed some carbonyl reductase had potential for industrial application in synthesis of(R)-3,5-BTPE,(S)-CFPL and(S)-2-chloro-1-(2,4-dichlorophenyl)ethanol.The carbonyl reductase KR01 from Leifsonia sp.S749 could bioreduce 3',5'-bis(trifluoromethyl)acetophenone [3,5-BTAP] into(R)-3,5-BTPE and 2-chloro-1-(3,4-difluoro-phenyl)-ethanone [CFPO] into(S)-CFPL with excellent activity and enantioselectivity.The carbonyl reductase LK05 from Lactobacillus kefir could convert 2,2',4'-trichloroacetophenone into(S)-2-chloro-1-(2,4-dichlorophenyl)ethanol with excellent activity and enantioselectivity.Secondly,in order to enhance conversion efficiency at high substrate concentration,the reaction conditions were optimized by response surface analysis.The result showed that(R)-3,5-BTPE(>99.9% ee)was synthesized from 600 g/L 3,5-BTAP with 98.3% yield and 230.26 mmol/L/h space–time yield under the optimized reaction conditions by E.coli/pET-28a(+)-KR01 whole cells.In addition,the E.coli/pET-28a(+)-KR01 recombinant E.coli cells could be repeatedly used up to 7 times in the reaction mixture containing 90% isopropanol.This is highest substrate loading and productivity for bioreduction of 3,5-BTAP by carbonyl reductase ever reported,which supplies a highlevel and cost-effective process for production of(R)-3,5-BTPE.Finally,the optimized reaction conditions were investigated in pilot scale for production of(S)-CFPL.When pH is 6.5,it can not only efficiently synthesize(S)-CFPL,but also reduce impurities.Within 4 hours and 24 hours,the E.coli/pET-28a(+)-KR01 recombinant cells could completely convert 150 g/L CFPO and 400 g/L CFPO into(S)-CFPL with excellent stereoselectivity(ee >99 %)and 196.8 mmol/L/h maximal space–time yield.It has been realized an industrial production of(S)-CFPL by E.coli/pET-28a(+)-KR01 with low production cost.