Cloning,Expression And Characterization Of Three 2,3-butanediol Dehydrogenase From Serratia Sp.T241

2,3-Butanediol(2,3-BD)is an important biobased platform chemical compound because of its potential application involved in the field of chemical engineering,food industry and biofuel.2,3-BD can be formed by many bacterial speices such as the genus of Klebisella,Serratia,Bacillus and Enterobacter.Generally,two of 2,3-BD isomers can be produced by these strains.However,depending on various BDHs differing in their stereospeciificities,the composition of 2,3-BD isomers produced by bacteria differs among different strains.In our previous study,a newly isolated strain designated as Serratia sp.T241 was found to produce three 2,3-BD isomers and exhibited high 2,3-BD yield.In this present thesis,to investigate the mechanism involved in three 2,3-BD isomers formation,we identified three 2,3-butanediol dehydrogenases from the T241 strain based on the Serratia sp.AS 12 genome sequence.Three genes encoding the three 2,3-butanediol dehydrogenase from the T241 strain were cloned and expressed in E.coli,and the recombinant enzymes were purified and characterized for enzymatic properties.The detailed work was introduced as followed.1.Screening of candidate genes based on the Serratia sp.genome sequenceBased on the Serratia sp.AS 12 genome sequence from NCBI database,a search was carried out using the protein sequences of meso-BDH,S,S-BDH,and R,R-BDH from Serratia marcescens,Rhodococcus erythropolis and Paenibacillus polymyxa,respectively.Three protein sequences from sp.AS 12 sharing 95%,64%,and 62%identities with those of meso-BDH,S,S-BDH,and R,R-BDH were found.Further sequence analysis showed the three genes contained 756bp,786bp and 1080bp,encoding 25 1aa,261aa and 359 aa respectively.2.Cloning,expression and catalytic properties of meso-BDHPCR-amplification was carried out by the primers designed according to the above obtained meso-BDH sequence.Sequencing results revealed that the amplified sequence shared 99%identity with that of sp.AS 12,suggesting that the amplified sequence was correct.So the meso-BDH gene was cloned into pET28a,and expressed in E.coli induced by IPTG.The recombinant meso-BDH purified using Histrap Kit exhibited the ability to catalyze the interconversion of R-acetoin/meso-2,3-BD and S-acetoin/S,S-2,3-BD.Additionally,when diacetyl was used as a substrate with the enzyme,S-acetoin could be formed.While R,R-2,3-BD is not a substrate at all for meso-BDH.3.Cloning,expression and catalytic properties of SyS-BDHPCR-amplification was performed by the primers designed according to the above obtained S,S-BDH sequence.Sequencing results revealed that the amplified sequence shared 97%identity with that of Serratia sp.AS 12,suggesting that the amplified sequence was correct.So the S,S-BDH gene was cloned into pET28a,and expressed in E.coli induced by IPTG The recombinant S,S-BDH purified using Histrap Kit exhibited absolute specifity from diacetyl to S,S-2,3-BD via S-acetoin.Additionally,S,S-2,3-BD could be also converted into S-acetoin by the S,S-BDH enzyme.While meso-2,3-BD,R,R-2,3-BD and R-acetoin were not substrates at all for S,S BDH.4.Cloning,expression and catalytic properties of R-BDHPCR-amplification was performed by the primers designed according to the above obtained sequence.Sequencing results revealed that the amplified sequence shared 97%identity with that of Serratia sp.AS 12,suggesting that the amplified sequence was correct.So the R,R-BDH gene was cloned into pET28a,and expressed in E.coli induced by IPTG The recombinant purified using Histrap Kit exhibited the ability to catalyze the interconversion of R-acetoin/R,R-2,3-BD and S-acetoin/meso-2,3-BD.Additionally,when diacetyl was used as a substrate with the enzyme,R-acetoin could be formed.While S,S-2,3-BD is not a substrate at all for R,R-BDH.Based on the above research results,we infered that meso-2,3-BD was formed by meso-BDH and R,R-BDH.While meso-BDH and contributed the formation of S,S-2,3-BD and from S-acetoin and R-acetoin respectively.Further research by inactivation of the three genes is necessary for confirmation of their function.