Mechanism Of 2,3-butanediol Stereoisomers Formation In Serratia Sp.T241 And Its Application

2,3-Butanediol(2,3-BD)and its dehydrogenation product,acetoin(AC),are important platform compounds.2,3-BD contains two stereo centers,resulting in three stereo isomers including(2R,3R)-,meso-and(2S,3S)-forms,and AC exists in two stereoisomeric forms:(3R)-acetoin and(3S)-acetoin,which are important potential pharmaceutical intermediatesSerratia sp.T241,a newly isolated 2,3-BD producing strain in our lab,could utilize sucrose and glucose as carbon sources to produce 2,3-BD.Interestingly,the strain T241 could produce three isomers of 2,3-BD simultaneously during the sugar fermentation process.Usually natural microorganisms produce a mixture of(2S,3S)-2,3-BD/meso-2,3-BD or(2R,3R)-2,3-BD/meso-2,3-BD.It suggested that there might be more complicated mechanism of AC and 2,3-BD stereoisomers formation in Serratia sp.T241.In this present thesis,four putative genes involved in biosynthesis of 2,3-BD isomers were identified.In order to investigate the mechanism involved in three 2,3-BD isomers formation,the obtained putative BDH/GDH genes were selected to perform in vitro and in vivo experiments.Furthermore,gene deletion and enzyme assay verified the roles of the genes for AC and 2,3-BD isomers formation in Serratia sp.T241.Finally,on the basis of mechanism confirmed in this thesis,the biotransformation of meso-2,3-BD into(3R)-acetoin using whole-cell biocatalytic method was investigated.Additionally,the coupled whole-cell biocatalytic system was developed for optically pure(2S,3S)-2,3-BD production,and application of immobilized cells as biocatalysts makes the whole-cell biocatalytic system recyclable and reusable.The detailed work was introduced as followed:1.Mechanism of 2,3-butanediol stereoisomers formation in Serratia sp.T241In this study,four genes(bdhl,bdh2,bdh3 and gdh)involved in AC and 2,3-BD isomers formation in Serratia sp.T241 were identified,and their deduced amino sequences shared high identities of 95%,64%,68%and 91%with meso-BDH from S.marcescens H30,(2S,3S)-BDH from R.erythropolis,(2R,3R)-BDH from B.subtilis 168 and GDH from S.marcescens H30.Additionally,the conserved domain of the BDH/GDH enzyme was investigated by using InterProScan web server.The results indicated that meso-/SS-BDH and RR-BDH belong to the SDR superfamily and MDR superfamily,respectively,whereas GDH belongs to the type ? Fe-ADH superfamily.Furthermore,the four genes encoding the BDH1,BDH2,BDH3 and GDH enzymes was cloned and expressed in E.coli BL21(DE3),purified and characterized.The results showed that,all the four purified enzymes exhibited the activities for AC and 2,3-BD as substrates.The main differences occurred in 2,3-BD oxidation reaction.(2S,3S)-2,3-BD and meso-2,3-BD were the substrates of BDH1 and BDH2,which showed no activity for(2R,3R)-2,3-BD.While BDH3 and GDH showed the activities for(2R,3R)-2,3-BD and meso-2,3-BD as substrates,(2S,3S)-2,3-BD was not the substrate for BDH3 and GDH at all.According to their Km and Kcat values,the four enzymes(BDH1,BDH2,BDH3 and GDH)should be categorized as meso-BDH,(2S,3S)-BDH,(2R,3R)-BDH and GDH.Furthermore,in vitro conversion and in vivo assemble of 2,3-BD pathway in E.coli revealed that the four enzymes contributed to meso-2,3-BD production in Serratia sp.T241.(2S,3S)-2,3-BD was obtained from(3S)-AC by BDH1 and BDH2,whereas BDH3 and GDH led to(2R,3R)-2,3-BD formation from(3R)-AC.The detailed model of AC and 2,3-BD isomers formation by Serratia sp.T241 could be inferred from the data of stereospecificity in catalytic reaction by the four purified enzymes.(3R)-AC was produced from pyruvate via ?-acetolacetate by ALS and ALDC,and DA was formed by a non-enzymatic oxidation decarboxylation of ?-acetolactate.In Serratia sp.T241,(3S)-AC production from DA was afforded by BDH1,BDH2 and GDH,whereas BDH3 led to(3R)-AC formation from DA.BDH1 and BDH2 exhibited high(S)-enantioselectivity for AC as substrate and catalyzed(3S)-AC and(3R)-AC into(2S,3S)-2,3-BD and meso-2,3-BD,respectively.In contrast,high(R)-enantioselectivity of BDH3 and GDH led to(2R,3R)-2,3-BD and meso-2,3-BD production from(3R)-AC and(3S)-AC.Futhermore,the four enzymes showed the abilities in the conversion from 2,3-BD to AC,and the corresponding configuration of AC could be obtained.It suggested that BDH1,BDH2,BDH3 and GDH were four reversible enzymes for the interconversion between AC and 2,3-BD.In addition,no DA could be detected from any form of 2,3-BD and AC,which showed that the conversions in catalytic reactions by the four enzymes were irreversible between DA and AC.In vitro conversion showed that BDH1,BDH2,BDH3 and GDH have the abilities in the interconversion between AC and 2,3-BD.However,the actual transcriptional expression levels of the four enzymes and whether they perform the corresponding function during the 2,3-butanediol fermentation in Serratia sp.T241 was not confirmed.Therefore,four mutants deficient in BDH1,BDH2,BDH3 and GDH were constructed.The results showed that ?bdh1 led to reduced concentration of meso-2,3-BD and(2S,3S)-2,3-BD by 97.7%and 87.9%.(2R,3R)-2,3-BD with a loss of 73.3%was produced by ?bdh3.Enzyme activity assays showed the decrease of 98.4%and 22.4%by ?bdh1 and?bdh3 compared with the wild strain.It suggested BDH1 and BDH3 played important roles in 2,3-BD formation,BDH2 and GDH have small effects on 2,3-BD production by Serratia sp.T241.2.Biosynthesis of(3R)-Acetoin from meso-2,3-Butanediol by whole cell of recombinant E.coli BL21(DE3)/pET28a-mbdh-nox-vgbPrevious studies showed that optically pure AC could be used as a precursor to synthesize novel optically active materials such as?-hydroxyketone derivatives and liquid crystal composites.Moreover,it was shown that(3R)-AC was a female sex pheromone,which can attract swarming males.However,the fermentation products of natural AC producing strains are racemic mixture,which limits the application of acetoin.In this study,whole cell of engineered E.coli with co-expression of meso-2,3-BDH and NADH oxidase was used for(3R)-AC production from meso-2,3-BD.Biotransformation conditions including pH,temperature,WCW,substrate concentration,metal ions,and reaction volume were optimized in shake flask.A high(3R)-AC yield of 86.74 g/L was obtained under the optimal conditions.In the optimization process,we accidentally found that oxygen which as substrate for cofactor regeneration significantly affected the(3R)-acetoin synthesis.Therefore,the gene vgb encoding Vitreoscilla hemoglobin(VHb)was introduced into the recombinant E.coli for improving oxygen supply during the whole-cell biocatalytic process.The presence of vgb and production of VHb resulted in an increase of(3R)-AC yield.Ultimately,86.74 g/L of(3R)-AC with the productivity of 3.61 g/L/h and the stereoisomeric purity of 97.89%was achieved from 93.73 g/L meso-2,3-BD using batch biotransformation in 5 L bioreactor.3.Repeated production of(2S,3S)-2,3-BD by an immobilized whole-cell biocatalyst(2S,3S)-2,3-BD has important application value.However,the precursor of(2S,3S)-2,3-BD is diacetyl(DA),an intermediate of 2,3-BD fermentation produced by the non-enzymatic oxidation of ?-acetolacetate.Due to the low concentration of DA in microbial fermentation,(2S,3S)-2,3-BD can only be produced at low concentrations.Additionnaly,the toxicity of DA on cells and enzymes was found to limit the biotransformation process.Luckily,our previous studies showed that(3S)-AC,which is also the precursor of(2S,3S)-2,3-BD,could be obtained from meso-2,3-BD by reversible enzyme(2R,3R)-2,3-BDH with NAD+as a coenzyme.Therefore,meso-2,3-BD is a suitable candidate as substrate for(2S,3S)-2,3-BD production due to its nontoxic feature and extensive source.In this study,the biotransformation of meso-2,3-BD into(2S,3S)-2,3-BD was investigated using coupled whole-cell system.We used whole E.coli cells co-expression of(2R,3R)-2,3-BDH and NADH Oxidase for the production of(3S)-AC from meso-2,3-BD.Furthermore,whole cell of engineered E.coli with co-expression of(2S,3S)-2,3-BDH and formate dehydrogenase was used for(2S,3S)-2,3-BD production from(3S)-AC.Biotransformation conditions including pH,temperature,WCW,substrate concentration,and metal ions were optimized in shake flask using free whole cells.As the results showed,the conversion of meso-2,3-BD into(2S,3S)-2,3-BD was achieved 92.5%with stereoisomeric purity of 96.2%when the substrate concentration was lower than 40 g/L.Whereas the stereoisomeric purity is not ideal when the substrate concentration is greater than 40 g/L.In order to take into account the product concentration and stereoisomeric purity,the couple system containing E.coli/pET-rrbdh-nox and E.coli/pET-ssbdh-fdh was immobilized individually using calcium alginate.For the preparation of beads with the proper permeability and rigidity,parameters such as the sodium alginate concentration,and the CaCl2 concentration were optimized.Furthermore,the reusability of free and immobilized whole cells was investigated.As the results showed,the immobilized whole-cells retaining their relative productivity at 81.6%after 3 cycles of successive re-use.In contrast,the free whole-cell system lost 74.2%of its ability to produce(2S,3S)-2,3-BD after 3 cycles.