Heterologous Expression,Directed Evolution,and Its Application Of Carbonyl Reductase Derived From Leifsonia Xyli HS0904

Chiral alcohols are a group of the most important building blocks,and have been widely used in the pharmaceutical,chemical and agriculture industries for production of chiral information into the products.The asymmetric reduction of prochiral carbonyl compounds is an effective approach for the preparation of optically active alcohols by oxidoreductases with high stereoselectivity or their whole cells as biocatalysts.Carbonyl reductase belongs to short chain dehydrogenases/reductases family,and catalyzed NAD(P)H-dependent biocatalytic asymmetric reduction with highly chemo-,regio-and enantioselectivity.Therefore,the development of novel and robust efficient carbonyl reductase is of important theoretical and practical significance for enriching microbial reductase library,exploreing the molecular catalytic mechanism and industrial application.(R)-3,5-bis(trifluoromethyl)phenyl ethanol((R)-BTPE)is a key chiral intermediate for the synthesis of Aprepitant(Emend(?))and Fosaprepitant(Ivemend(R)),a tachykinin NK1 receptor antagonist widely administered to cancer patients without the nausea and vomiting side effects that often happen with chemotherapy.The biocatalytic asymmetric reduction of 3,5-bis(trifluoromethyl)acetophenone(BTAP)is an effective approach for the preparation of(R)-BTPE.In this dissertation,biocatalytic asymmetric reduction of BTAP to(R)-BTPE was used as model reaction,the carbonyl reductase derived from Leifsonia xyli HS0904 was employed as biocatalyst,and its characterization,catalytic function and the structure-activity relationships were explored by enzymatic purification,gene cloning,directed evolution,as well as molecular simulation.Our research findings provide a new enzyme source and new way for biocatalytic preparation of functional chiral compounds,which is of great importance for better understand the catalytic function and molecular catalytic mechanism of LXCAR.Firstly,a L.xyli HS0904 derived carbonyl reductase(LXCAR)was purified and characterized.The purified LXCAR was obtained from the cell-free extract by ammonium sulfate precipitation,weak-anion exchange chromatography on DEAE-sepharose FF column,hydrophobic interaction chromatography on octyl-sepharose 4 FF column,strong-anion exchange chromatography on High Q column and gel filtration on SuperdexTM 200 column,and showed a single band(about 24 kDa)on SDS-PAGE.The molecular mass of the purified LXCAR was estimated to be 49 kDa by a SuperdexTM 200 column,thus the LXCAR might be a homologous dimer protein comprising subunits of identical molecular weight.The N-terminal amino acid sequence of the LXCAR was determined to be AQYDVAGRSAI.The purified LXCAR exhibited a maximum enzyme activity at 34? and pH 7.2,and retained over 90%of its initial activity at 4? and pH 7.0 for 24 h.The purified LXCAR were activated by Ca2+,Mg2+,Mn2+,Co2+,L-cysteine,L-glutathione,urea,PEG 1000 and PEG 4000,while inhibited by Sn2+,Fe2+,Pb2+,Fe3+,EDTA,isopropanol,DMF,tetrahydrofuran and n-hexane.The Vmax and Km of the purified LXCAR for BTAP and NADH were confirmed as 33.9 U/mg,0.383 mM and 69.9 U/mg,0.412 mM,respectively.Furthermore,the LXCAR was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.Then,gene cloning and directed evolution of the LXCAR were carried out,and its molecular catalysis mechanism was also explored using molecular simulation methods.Based on the N-terminal amino acid sequence of native LXCAR and its results for flight mass spectrometry and homologous sequence alignment,a series of degenerate primers were designed.The LXCAR gene was successfully cloned from the genomic DNA of L.xyli HS0904,and overexpressed in Escherichia coli BL21(DE3).Recombinant E.coli crude extract showed a specific activity of 1.54 U/mg,which was 62 times higher than that of L.xyli HS0904 crude extract.Bioinformatics analysis indicated that the amino acid sequence of the LXCAR showed 89%similarity to short-chain dehydrogenase/reductase from L.sp.S749(LSADH),and two consensus sequences of the short-chain dehydrogenase/reductases(SDRs)family,an N-terminal TGX3GXG cofactor-binding motif and a SX12YX3K segment which is essential for catalytic activity of SDRs,are fully conserved in the LXCAR.Moreover,the LXCAR is further found to be a dimer protein comprised of two identical subunits of 24 kDa,and different from the LSADH involved in L.sp.S749 which was reported to be consisted of four identical subunits of 25 kDa,thus belongs to a new member of the SDRs family.The LXCAR was engineered by using error-prone polymerase chain reaction and site-directed mutagenesis,and a mutant with high reduction activity(LXCAR-S154Y)was obtained.Sequence analysis indicated that tyrosine residue replaces the original serine residue at 154 in the LXCAR-S154Y.The LXCAR-S154Y was purified and characterized,and exhibited nearly 2.3-fold,5.4-fold and 12.6-fold increase in specific activity,kcat value and kcat/Km value toward BTAP,respectively,compared with the LXCAR.Three-dimensional structure of LXCAR-S154Y was built using Discovery Studio 3.0 based on the crystal structure of the SDR from Sinorhizobium meliloti 1021 and oxidoreductase from Thermus thermophilus HB8 in complex with NADH,which have the highest sequence similarity to the LXCAR-S154Y in the PDB entries.The LXCAR-S154Y structure was confirmed that the quality of the model is legitimate by Ramachandran plot using PROCHECK program.Docking studies of the LXCAR-S154Y complex with BTAP was carried out by flexible docking using Discovery Studio 3.0 to understand the combination relationship between enzyme and substrate and explore its molecular catalysis mechanism for asymmetric reduction.The results showed that the carbonyl oxygen atom of BTAP forms hydrogen bonds with Tyr157 and Ser144,and maintains very close to Tyr157,possibly because the carbonyl oxygen atom of the substrate is protonated from the Tyrl57-OH which as a hydrogen donor,thus it is more effective for hydroxylation of the substrate by the attacking of the hydride from the C4 atom of nicotinamide ring of NADH to the carbonyl carbon atom;Lys161 makes a hydrogen bond with the O2' atom of the nicotinamide mononucleotide ring of the cofactor,possibly due to Lys161 as a hydrogen donor provide a hydrogen atom to the O2' atom,then the O2' atom donates hydrogen atoms to the OH atom of Tyr157,thus Tyrl57-OH is regenerated;Asn116,which is close to Lys161 residue,while being in contact with the solvent may act as a transport system to transfer new protons from the bulk solvent to the active center allowing the reprotonation of the Lys 161.Additionally,the conformations of the LXCAR and LXCAR-S154Y complex with BTAP were analyzed,and the binding energies of the BTAP/LXCAR-S154Y complexes(-79.3 kcal/mol)was lower than those of the BTAP/LXCAR complexes(-10.3 kcal/mol),and the distances were 3.99 and 3.78 A between the C4 atom and the carbonyl carbon in the conformations of the LXCAR and LXCAR-S154Y complex with BTAP,respectively.These results suggested that the LXCAR-S154Y/BTAP complexes is more stable than the LXCAR/BTAP complexes,and substrate is closer to the nicotinamide mononucleotide ring of the cofactor when forming enzyme-substrate complexes as a result of the space structure of Tyr is bigger than Ser,thus it is easier for hydroxylation of the substrate by the attacking of the hydride from the C4 atom of nicotinamide ring to the carbonyl carbon atom.Finally,glucose fed-batch cultivation of the recombinant E.coli was carried out,and the biocatalytic process of the asymmetric reduction of BTAP to(R)-BTPE in buffer system and ionic liquids-based buffer system by recombinant E.coli were also investigated.The results indicated that the maximum biomass and enzymatic activity were 32.4 g/L(dry cell weight(DCW))and 308.5 ?mol/min/gcell,respectively,during the process of glucose fed-batch cultivation.Moreover,the process parameters for the asymmetric reduction of BTAP to(R)-BTPE in buffer system were investigated,and the optimal isopropanol concentration,reaction temperature,buffer pH value,substrate concentration and cell concentration were 20%(v/v),30?,7.2,1000 mM and 12.7 g/L(DCW),respectively.The asymmetric reduction of BTAP to(R)-BTPE was also successfully conducted in a novel[N1,1,1,1][Cys]-based ionic liquid buffer system by recombinant E.coli,and the optimum[N1,1,1,1][Cys]content,buffer pH,reaction temperature,substrate concentration and cell concentration for the bioreduction were 3.5%(w/v),6.8,30?,1000 mM and 12.7 g/L(DCW),respectively.Under the optimized conditions,the recombinant E.coli cells was found to asymmetrically catalyze the reduction of BTAP to(R)-BTPE at 1000 mM BTAP(256 g/L)with 98.7%of product yield and above 99%of product ee in the[N1,1,1,1][Cys]-containing buffer system.To our knowledge,this is the highest substrate loading and product yield reported so far for the biocatalytic asymmetric reduction of BTAP to(R)-BTPE.The biocatalytic process for the asymmetric reduction of BTAP to(R)-BTPE in buffer system with and without[N1,1,1,1][Cys]on a 5.0 L preparative scale were also investigated,and the best yield were 93.9%and 80.9%within 12 h,respectively.Moreover,the product ee were both above 99%.Additionally,products were separated from reaction mixture by n-hexane extraction,vacuum distillation and silica gel column chromatography,and then identified by GC analysis,1H NMR,13C NMR and polarimetric analysis.The results indicated that the purity of product was above 99%,and the product was identified as(R)-BTPE.