Excavation Of Noval Ketoreductases From Paraburkholderia Phymatum STM815 And Its Application In The Synthesis Of(R)-2-Chloro-1-(3,4-difluorophenyl)Ethanol

In recent years,the market for chiral drugs has grown rapidly.As the key intermediates of chiral drugs,the synthesis methods of chiral alcohols have received more and more attention.Compared with the traditional chemical methods,the biocatalytic asymmetric reduction method has high catalytic efficiency,strong stereoselectivity,mild reaction conditions,and environmental friendliness,making it an effective way to synthesize chiral alcohols.(R)-2-chloro-1-(3,4-difluorophenyl)ethanol[(R)-CFPL]is an important chiral intermediate for the synthesis of ticagrelor(an anticoagulant drug for the treatment of acute coronary syndrome).In this study,we focused on mining and screening of novel ketoreductase genes,using co-expression technology and reaction process engineering to establish an efficient asymmetric synthesis process of(R)-CFPL,and the obtained ketoreductase was applied in the efficient synthesis of a series chiral alcohols.The specific research contents are as follows:(1)Excavation and screening of novel ketoreductases.52 potential ketoreductase genes were excavated from Paraburkholderia phymatum STM815,and successfully cloned and expressed heterologously.The glucose dehydrogenase from Bacillus subtilis and the candidate ketoreductase were used to construct a coenzyme recycle system to perform asymmetric reduction of 2-chloro-1-(3,4-difluorophenyl)ethanone(CFPO).A noval enzyme PpKR8 was screened with high activity and high stereoselectivity(ee>99.9%)in catalyzing the synthesis of(R)-CFPL.Multiple sequence alignment showed that PpKR8 is a short-chain dehydrogenase,and has low similarity(31-34%)to the reported short-chain dehydrogenases with certain activity on CFPO,which has potential research value.(2)Characterization on enzymatic properties of PpKR8.The results show that:the optimal reaction temperature of PpKR8 is 40℃,while the thermal stability is better at 35℃;the optimal reaction pH is pH 6.0 in phosphate buffer,and it is more stable in a neutral and acidic environment;the addition of 5%(v/v)DMSO greatly improved the conversion efficiency;PpKR8 has high affinity and catalytic efficiency for CFPO,the Km value and kcat/Km value are 0.85 mM and 1.9×102 mM-1 s-1,respectively;PpKR8 has a wide substrate spectrum,showing catalytic activity to 35 kinds of α/β-ketoesters,aromatic ketones and heterocyclic ketones,especially for ethyl 4-chloroacetoacetate,3’-chloroacetophenone and N-boc-3-piperidone with activity of 290 U mg-1,253 U mg-1,and 215 U mg-1,respectively.(3)Construction of co-expression system.In order to reduce the mass transfer limitation,reduce the complexity and cost of the reaction,a co-expression strain was constructed to realize the heterologous co-expression of PpKR8 and BsGDH in E.coli.The enzyme activity assay proved that the co-expressing PpKR8-BsGDH dry cells demonstrated a reducing activity of 216.9 U g-1 and an oxidizing activity of 570.4 U g-1,retaining most of the biocatalytic activity compared to the corresponding single-expressing dry cells.Compared with cell-coupled system,the co-enzyme transfer efficiency in co-expression system is higher and the reaction period is only a half of the cell-coupled system.(4)Establishment of biocatalytic asymmetric reduction process.In order to establish an economically feasible and efficient process path for the preparation of(R)-CFPL,the required substrate concentration,biocatalyst loading,and cofactor dosage were optimized.Finally,under the conditions of 15 g/L dry cells and 0.1 mM NAD+,1.57 M(300 g/L)CFPO could be completely transformed into(R)-CFPL in 24 hours with a turnover number(mol/mol)of 15473,which was the highest value reported in the literature so far.(5)Application of PpKR8 in the synthesis of other chiral alcohols.In order to expand the industrial application range of PpKR8,the co-expression system was used to biocatalyze the asymmetric synthesis of several other chiral alcohols used as important drug intermediates(R)-ethyl-4-chloro-3-hydroxybutanoate,(R)-2-chloro-1-(2,4-dichlorophenyl)ethanol,and(S)-3-hydroxy-1-boc-piperidine.It was found that PpKR8 could catalyze the asymmetric synthesis of 333 g/L(2 M)(R)-CHBE,226 g/L(1 M)(R)-CPEO,and 151 g/L(0.75 M)(S)-NBHP in 10-18 h with>99.9%ee value,demonstrating a relatively broad application prospects.