| 2,3-Butanediol is a platform and fuel biochemical that can be efficiently produced from biomass.Until now,a number of microorganism species can be used for the microbial production of 2,3-butanediol,such as,Klebsiella,Enterobacter,Bacillus and Serratia.The biosynthesis pathway of 2,3-butanediol aready has been studied exensively,there are three key enzymes in bacterial 2,3-butanediol formation pathway,a-acetolactate synthase(ALS),a-acetolactate decarboxylase(ALDC),and 2,3-butanediol dehydrogenase(BDH,also known as acetoin reductase,AR).In bacterial metabolism,firstly,carbohydrate generally be converted to pyruvate through the glycolytic pathway.Two moleculers of pyruvate can be transformed into a-acetolactate catalyzing by a-acetolactate synthase,following,it can be converted into acetoin catalyzing by a-acetolactate decarboxylase.2,3-butanediol dehydrogenase catalyze reversible reaction from acetoin to 2,3-butanediol,with the form of NAD(NADP)changing.There are three stereoisomer of 2,3-butanediol:meso-,(2R,3R)-,(2S,3S)-.Microbial 2,3-butanediol production generally generate a mixture of two stereoisomers.We cloned the 2,3-butanediol dehydrogenase of aco gene cluster and expressed it in E.coli BL21(DE3)with the commercial plasmid pETDuet.Then,we purified the BDH with Ni-affinity chromatography and study the enzymatic properties.Also,we find two quinoprotein alcohol dehydrogenase,QedH818 and QedH823,which's cofactor is PQQ,the homologue of them in P.putida HK5 have enzyme activities of 2,3-butanediol,so we have the reason to infer the QedH818 and QedH823 can dehydrogenate 2,3-butanediol.In order to study the dehydrogenation mechanism of 2,3-butanediol in P.putida KT2440,we conduct the deletion of the three BDH.The mutant strain which lost all the three gene was unable to grow on 2,3-butanediol.By means of comparation of different course of P.putida KT2440 cultured on the three stereomer of 2,3-butanediol.The conclusion is that inexistence of 2,3-butanediol racemase and acetoin racemase may be exist.It need further experimental proof indeed.Although the microbial production of 2,3-butanediol is really high.However,a value-added process for this chemical has not yet been developed.To expand the utilization of 2,3-butanediol produced from biomass,an improved derivative process of 2,3-butanediol is desirable.In this study,a Gluconbacter xydans strain DSM 2003 was found to have the ability to transform.2,3-butanediol into acetoin,a high value feedstock that can be widely used in dairy and cosmetic products,and chemical synthesis.All three stereoisomers,meso-2,3-butanediol,(2R,3R)-2,3-butanediol,and(2S,3S)-2,3-butanediol,could be transformed into acetoin by the strain.After optimization of the bioconversion conditions,the optimum growth temperature for acetoin production by strain DSM 2003 was found to be 30? and the medium pH was 6.0.With an initial 2,3-butanediol concentration of 40 g/L,acetoin at a high concentration of 89.2 g/L was obtained from 2,3-butanediol by fed-batch bioconversion with a high productivity(1.24 g/L · h)and high yield(0.912 mol/mol).The results demonstrate that the method developed in this study could provide a promising process for efficient acetoin production and industrially produced 2,3-butanediol utilization.In this paper,we just study the dehydrogenation mechanism of 2,3-butanediol in P.putida KT2440,the decomposition of acetoin have not be mentioned.Although the yield of acetoin by utilizing G.oxydans DSM 2003 is really high,the related gene have not been charactered.The heterologous expression of 2,3-butanediol dehydrogenase with cofactor PQQ is not successed in this study. |
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