Cloning,Expression,and Application Of A Carbonyl Reductase From Burkholderia Cenocepacia

(R)-3,5-bis?trifluoromethyl?phenyl ethanol??R?-BTPE?is an important chiral intermediate for the synthesis of aprepitant,which can be produced by asymmetric reduction of the prochiral ketone 3,5-bis?trifluoromethyl?acetophenone?BTA?.In this study,a carbonyl reductase?WP₀12492403.1?derived from Burkholderia cenocepacia was discovered with excellent anti-Prelog stereoselectivity of reducing BTA,and ee values at>99.9%.Bc-3 is a homotetramers with 247 resuides long,which belong to the short-chain dehydrogenase/reductases family,and 3D structure display a typical sequence motifs,comprising Rossmann-fold elements for coenzyme-binding and the highly conserved catalytic tetrad of Ser-Tyr-Lys.The optimal reaction temperature and pH of asymmetric reduction of BTA by using whole-cell of Bc-3 recombinant strain was 25 ? and pH7.0,respectively.The activity of Bc-3 was shown an enhancement in the presence of most metal ions except for Cu2+ and Ni2+.In addition,it was observed that the relative activity of Bc-3 towards acetophenone and 2'-fluoroacetophenone were 119.5%and 200.1%,respectively.Synthesis of ezetimibe requires enantiopure 3-[5-?4-fluorophenyl?-5?S?-hydroxypentanoyl]-4?S?-4-phenyl-1,3-oxazolidin-2-one??S?-FOP alcohol?as a crucial intermediate which is produced by reduction of 1-?4-fluorophenyl?-5-?2-oxo-4-phenyl-oxazolidin-3-yl?-pentane-1,5-dione?FOP dione?.we have investigated the effect of 142 mutants and 185 mutants on the asymmetric reduction of BTA and FOP dione.N142S,Y185C,Y185F,Y185N and N142S/Y185C were detrimental to the reduction of BTA,which has apparent negative effect on the activity and enantioselectivity.In contrast,they showed a stronger enhancement of the enzyme activity towards FOP dione.The conversion of FOP dione by N142S/Y185C mutant was increased to 22.0%,and Y185C,Y185F and Y185N mutants has high diastereomeric selectivity for FOP dione,with de value>99.9%.In addition,Komagataella pastoris GS11 was discovered with high diastereomeric selectivity for the asymmetric reduction towards FOP-dione.