Cloning Of The Gene Encoding Carbonyl Reductase And Its Application Of Biological Preparation For Duloxetine Key Intermediate

Duloxetine is a 5-serotonin(5-HT)and norepinephrine(NE)reuptake dual inhibitors for treating severe depression.(S)-N,N-dimethyl-3-hydroxy-(2-thiophene)-1-propylamine((S)-DHTP)is the important chiral intermediate for the synthesis of duloxetine.Carbonyl reductase,a kind of the NADPH or NADH depended short chain dehydrogenase/reductase,can catalyze the prochiral ketones to optically active alcohols.In this paper,the reaction characteristics about reduction of DKTP in aqueous phase system were studied.The genes encoding carbonyl reductase were cloned from Kluyveromyce marxianus CGMCC 2.1977 and the recombinant strains for synthesis of the chiral drug intermediates were constructed.Firstly,7 kinds of relative strains were screened for asymmetric reduction(DKTP to(S)-DHTP)and the result showed that Kluyvermyce marxianus CGMCC 2.1977 had the best transformation effect,and that the conversion rate and enantiomeric excess(ee)were 72%and 99.5%respectively.Secondly,based on bioinformatics analysis of existing carbonyl reductase genes,carbonyl reductase genes from Kluyvermyce marxianus CGMCC 2.1977 were cloned using homology-based degenerate PCR.They were inserted into pMD 19-T Simple Vector and sequenced,the results show that the size of the two sequences are 1038 bp and 1026 bp respectively and 100%homology with the genes which login number are AB183149 and FR854195.1 in the Genbank database.And the two cloned genes are named cmcr and mcrf.Thirdly,cmcr and mcrf were cut from those T-A cloning vectors using restriction Enzymes and inserted into expression vector pET-28a(+).pET28a-CMCR and pET28a-MCRF which contain cmcr or mcrf were constructed.They were introduced into 2 kinds of Escherichia coli(BL21(BE3)and Rosetta(DE3))to express the enzyme respectively.The SDS-PAGE electrophoresis analysis manifested that when the host cells was Rossetta(DE3),the CMCR was overexpressed with a protein molecular weight of 42 KD while MCRF is intracellular insoluble protein.Subsequently,the biochemical characterization,the substrate specificity and coenzyme dependence of CMCR were investigated.The optimal temperature and pH for enzymatic reactions are 40 ℃ and 8 respectively.The enzyme has low thermal and pH stability.Ca2+ activates the enzyme activity obviously,especially when its concentration is 0.5 mmol/L.Finally,The asymmetric reduction of DKTP to(S)-DHTP)with Rosetta(pET28a-CMCR)cells as a catalyst was investigated,and e.e.value and transformation yield were 100%and 81.0%,respectively.The application of the cells to the asymmetric reduction of 4-chloro-3-oxobutanoateethyl ester(COBE)to(S)-4-chloro-3-hydroxyl butanoateethyl ester((S)-CHBE)was also attempted.