The Discovery Of Carbonyl Reductases From Kurthia Gibsonii SC0312 And Its Application In The Asymmetric Synthesis Of (R)-1-phenyl-1,2-ethanediol

Chiral alcohols play crucial roles in the fields of chiral drugs,flavors,agrochemicals and liquid crystals.Biocatalytic reaction for the fabrication of chiral alcohols has attracted considerable attentions because of its several merits including high enantioselectivity,mild reaction conditions,green solvents and sustainability,etc.However,biocatalysts are also generally subjected to some demerits such as poor stabilities and difficult reuse;moreover,wild-type strains have a complex intracellular enzyme system,low expression levels of key enzymes,and rigorous culture conditions,all of which partly restrict their applications in biocatalytic reactions.Our research group previously isolated a bacterial strain(Kurthia gibosonii SC0312)from soil,which is capable of asymmetrically reducing 2-hydroxyacetophenone(HAP)to(R)-1-phenyl-1,2-ethanediol{(R)-PED}with high enantioselectivity.Based on the results,this study aimed to discover the key carbonyl reductases responsible for asymmetric reduction of HAP from Kurthia gibsonii SC0312 cells with own intellectual property rights by genetic engineering technique,and then construct the multi-enzyme nanodevice and a recombinant E.coli strain with cofactor regeneration to improve the reaction efficiency of(R)-PED biosynthesis.Eight key enzyme genes from Kurthia gibsonii SC0312 cells were predicted,then cloned into E.coli BL21(DE3)by genetic engineering technology.After inducing expression,the catalytic activity and enantioselectivity of the reduction of HAP catalyzed by the engineered E.coli strains were determined.A key enzyme Kg BDH catalyzing HAP to(R)-PED with high enantioselectivity was mined,which is a homodimer with 37.5 KDa,encoding 349 amino acid residues.Besides,it is a member of medium chain dehydrogenase superfamily and has typical coenzyme binding and zinc motifs.Studying on enzymatic characteristics showed that the recombinant Kg BDH performed good activity at p H 6.0-8.0 and the highest activity at 45?,and preferable stabilities at p H 7.5-8.0 and 20-30?.And common metal irons have no activation effect on the recombinant Kg BDH.Furthermore,the Km,kcat and specific activity of the recombinant enzyme for the reduction of HAP were 5.4 mmol/L,4.9 s^-1,and 6.7 U/mg,respectively,at p H 7.5 and 35?.A multi-enzyme nanodevice with cofactor regeneration was established to prepare(R)-PED based on Kg BDH,FDH(formate dehydrogenase)and self-assembly system Spy Catcher/Spy Tag.Firstly,the fusion enzymes Kg BDH-Spy Catcher(Kg BDH-SC)and FDH-Spy Tag(FDH-ST)with self-assembling pair were constructed on the basis of protein fusion technology.After in vitro self-assembly between the two fusion proteins,the co-immobilized enzymes Kg BDH-SC-ST-FDH-SiO₂ were obtained by chemical crosslinking under the design conditions,which was 2.9 times higher than that of the conventional random co-immobilized enzymes in catalytic activity and remained 49% of initial activity.Kg BDH-SC-ST-FDH-SiO₂ also presented excellent catalytic activity at p H 7.0-7.5 and 35?.Compared with the mixture of free enzymes,it has better p H and organic solvents stability.Moreover,the co-immobilized enzymes remained 52.5% of initial activity after eight cycles.Finally,the co-immobilized enzymes Kg BDH-SC-ST-FDH-SiO₂ could asymmetrically reduce HAP of 60 mmol/L to chiral(R)-PED in phosphate buffer,affording 83.9% yield and>99% ee.A sequential co-immobilized enzymes FDH-ST-SC-Kg BDH@SiO₂ were fabricated to prepare(R)-PED using crosslinking and protein self-assembly by adjusting the immobilized sequence of Kg BDH-SC and FDH-ST.Under the optimum immobilization conditions,FDH-ST-SC-Kg BDH@SiO₂ had the activity recovery of 50% and exhibited excellent catalytic performance under p H 7.0-7.5 and 35-40?.Compared to the mixture of free enzymes,the sequential co-immobilized enzymes showed better p H stability and organic solvents tolerance.Additionally,the sequential co-immobilized enzymes for the reduction of HAP maintained 51.7% of initial activity after eight cycles.Ultimately,FDH-ST-SC-Kg BDH@SiO₂ could asymmetrically reduce HAP of 80 mmol/L to chiral(R)-PED in phosphate buffer,affording87.4% yield and>99% ee.A recombinant E.coli BL21(DE3)-p ETduet1-Kg BDH-GDH was constructed by co-expressing Kg BDH and GDH(glucose dehydrogenase).The optimum expression conditions of the recombinant strain were 0.1 mmol/L IPTG,20?,and 18 h.To improve the stability of the free cells,we used the mixture of activated carbon and calcium alginate to embed the recombinant E.coli engineered strains.The results showed that the reduction activity of HAP catalyzed by the cells immobilized by the mixture of activated carbon and calcium alginate was significantly higher than that of the cells immobilized by calcium alginate and was comparable to that of the free cells.The cells immobilized by the mixture of activated carbon and calcium alginate showed the highest activity for HAP reduction at p H 7.5 and performed good stabilities at p H 6.0-9.0 and 25-30?.Furthermore,with the superiority of operational stability to free cells,the immobilized cells for the HAP reduction remained 55% of initial activity after four batches.Finally,the immobilized cells can asymmetrically catalyze HAP of 240 mmol/L to(R)-PED of 195 mmol/L in 12 h by a fed-batch strategy,with 81% yield and>99% ee.