Identification And Application Of The Key Genes Involved In Acetoin And 2,3-butanediol Formation By S.Marcescens H30

2,3-Butanediol(2,3-BD)and its dehydrogenation product,acetoin(AC),are important platform compounds due to its potential applications involved in the field of chemical engineering and food industry.2,3-BD contains two stereo centers and has three stereoisomers including(2R,3R)-,meso-and(2S,3S)-forms,and AC exists in two stereoisomeric forms:(3R)-AC and(3S)-AC,which are important potential pharmaceutical intermediates.In this present thesis,the mechanism involved in AC and 2,3-BD formation by S.marcescens H30 was studied by using genetic engineering methods and modern biological technique.The detailed work was as followed:1.Enhancing 2,3-BD production by S.marcescens H30 with overexpressed BDHIn order to decrease the accumulation of AC and improve 2,3-BD production during the fermentation process,a constitutive expression vector pPbuK-BDH for over-expression of BDH was constructed,and the vector was transformed into S.marcescens H30 by electroporation,which resulted in S.marcescens H30-BDH.Batch fermentation in 5 L bioreactor showed that excess BDH could significantly decrease the AC accumulation by 86.1%,and increase 2,3-BD production by 78.5%.Meanwhile,the byproducts such as ethanol,lactate and succinate production were suppressed by 45.3%,27.2%and 51.9%due to over-production of BDH in S.marcescnes H30.Enzyme activity assay indicated that the activity of BDH in the recombinant strain showed over 21-fold higher when compared with wild strain,which resulted in a lower level of NADH pool and a higher level of NAD+.2.Cloning,expression,enzyme properties and catalytic properties of meso-BDHTo reveal the mechanism of AC and 2,3-BD isomers formation in S.marcescens H30,the BDH gene from S.marcescens H30 was cloned into pET28a,and expressed in E.coli induced by IPTG.The recombinant BDH purified using Histrap Kit exhibited the ability to catalyze meso-2,3-BD,(2S,,3S)-2,3-BD,(3S/3R)-AC and DA as substrates in the presence of NAD(H).While(2R,3R)-2,3-BD is not a substrate at all for BDH.The maximum activity and low Km value for meso-2,3-BD in the oxidation process were determined,and combined with the results from substrate specificity test,therefore,the BDH from S.marcescens H30 could be categorized as a NAD(H)-dependent meso-BDH.In the presence of NADH,meso-BDH could catalyze the reduction of DA and(3R)-AC to(3S)-AC and meso-2,3-BD,respectively,while(3S)-AC as a substrate could be further transformed into(2S,3S)-2,3-BD at pH 9.0.For diol oxidation reactions,(3R)-AC and(3S)-AC were obtained when meso-2,3-BD and(2S,3S)-2,3-BD were used as the substrates with meso-BDH and NAD+.In addition,the meso-BDH activity for meso-2,3-BD oxidation was enhanced in the presence of Fe2+ and for DA and(3S/3R)-AC reduction in the presence of Mg2+ and Mn2+,while several metal ions inhibited its activity,particularly Fe3r for reduction of DA and(3S/3R)-AC.3.Cloning,expression,enzyme properties and catalytic properties of GDHWhen we inactive the meso-BDH encoded by budC gene,it does not completely abolish 2,3-BD production,which suggests that another similar enzyme involved in 2,3-BD formation exists in S.marcescens H30.A GDH gene from S.marcescens H30 was cloned into pET28a,and expressed in E.coli induced by IPTG The recombinant GDH purified using Histrap Kit exhibited the ability to catalyze DA,(3S/3R)-AC meso-2,3-BD and(2R,3R)-2,3-BD.While(2S,3S)-2,3-BD is not a substrate at all for GDH.In the presence of NADH,GDH could catalyze the reduction of DA and(3R)-AC to(3S)-AC and(2R,3R)-2,3-BD,respectively,while(3S)-AC as a substrate could be further transformed into meso-2,3-BD.For diol oxidation reactions,(3S)-AC and(3R)-AC were obtained when meso-2,3-BD and(2R,3R)-2,3-BD were used as thesubstrates with GDH and NAD+.The enzyme exhibited relative high thermo tolerance with optimum temperature of 60? in the oxidation-reduction reactions.Additionally,the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe2+ and for(3S/3R)-AC reduction in the presence of Mn2+,while several cations inhibited its activity,particularly Fe2+ and Fe3+ for(3S/3R)-AC reduction.4.Metabolic engineering of E.coli for efficient production of(3R)-ACThe AC operon of S.marcescens H30 and the NADH oxidase gene(nox)from Lactobacillus brevis had been cloned in our laboratory.A synthetic pathway involved the budRAB and NADH oxidase gene in E.coli was developed for efficient AC production.Chiral-column GC analysis indicated that the stereo isomeric purity of AC produced was 97.3%.Furthermore,the fermentations conditions and medium composition were optimized in shake flasks by signal factor experiments and orthogonal design method.Under optimal conditions,(3R)-AC concentration reached 38.3 g/L in flask fermentation.Fed-batch fermentation based on a suitable agitation speed was carried out in a 5 L bioreactor,and maximum(3R)-AC concentration of 60.3 g/L was achieved with a productivity of 1.55 g/L-h and yield 86.3%.5.(3S)-AC production from DA by whole cell E.coli BL21(DE3)/pET28a-BDHThe effects of temperature,pH,cell density,auxiliary substrate concentration and substrate concentration on(3S)-AC production by the recombinant cells were investigated.Results showed that 37? was the optimal temperature and the pH of 8.0 was the highest rate.On that basis,the orthogonal experiment was performed.The optimal combination of catalysis were determined as follows:lOmL of the catalytic system,4.0 g/L of DA,1.8 g/L of the glucose,36 g/L of the bacteria wet weight,10 h of the catalytic time.Under the optimal conditions of biosynthesis of(3S)-AC the purity and the yield of(3S)-AC respectively were 98.11%and 3.51 g/L which increased 80.67%before optimization,and the transformation rate of DA was 87.75%.