Research On Mechanism Of 2,3-butanediol Production By Serratia Marcescens And Its Metabolic Engineering

As an jmportant biobased bulk chemical,2,3-butanediol has been shown to have potential applications in the manufacture of printing inks,perfumes,fumigants,moistening and softening agents,explosives and plasticizers,and as a carrier for pharmaceuticals.In this study,we not only cloned and characterized the genes involved 2,3-butanediol pathway in Serratia marcescens MG1,but also investigated how they are regulated by acetate and quorum sensing.Based on these,we constructed plasmids which can be applied to metabolic engineer the Serratia marcescens.All the obtained results should lead to strain improvement and more suitable fermentation strategy by using Serratia to produce 2,3-butanediol in industry.The detailed work was introduced as following:1)The cloning and characrizaiton of genes involved in 2,3-butanediol produced by Serratia marcescensFirst the partial sequence of the target gene was obtained by using degenerate primers,then upstream and downstream regions of the partial sequence was got by using genome walking technique.Combining these two methods,we successfully cloned the genes encoding a-acetolactate synthase(slaB),a-acetolactate decarboxylase(slaA),meso-2,3-butanediol dehydrogenase(slaC)and a regulator(slaR)belonging to the LysR family.The genes slaA,slaB and slaR locate in one operon,while the slaC gene exsits in anther position of the genome.Bioinformatics analysis showed that the strengths of them were 780bp(slaA),1686bp(slaB),756bp(slaC)and 1086bp(slaR),respectively,and encoded proteins of 260,561,251 and 361 residues.Their molecular weights and isoelectric points were 28.9kD,60.70kD,26.2kD,39.76kD and 5.38,5.89,5.39,9.12.Expression products of the genes with pET28a system exhibited comparable molecular weights using SDS-PAGE analysis.We then constructed mutants of these corresponding genes.The results showed that the slaA and slaB mutants were defective for acetoin and 2,3-butanediol production during fermentation.However,the inactivation of slaC can have great impact on 2,3-butanediol production.but it doesn't affect the production of aceotoin.In fact,that the slaC mutant could still produce a certain amount of 2,3-butanediol indicated the existence of other 2.3-butanediol dehydrogenases in Serratia marcescens.2)Research on the regulation of 2,3-butanediol production by Serratia marcescensAfter the genes involved in 2,3-butanediol biosynthesis were cloned and characterized,the following researches showed that they are dual regulated by the acetate and quorum sensing.Two proteins,SlaR and SwrR.exert positive and negative effects on this process,respectively.Acetate and the signal molecular BHL/HHL,act as inducing ligand of these two regulators,respectively.At low cell density.SwrR exerts efficient repression on the expression of the genes slaA and slaB.This effectively prevents acetoin production which subsequently results in no production of 2,3-butanediol.As the cell density increases,the repression effect of SwrR are gradually alleviated by the accumulation of BHL/HHL.At this point,enough acetate has accumulated triggering the transcription of genes slaA and slaB.Gene slaC,which isn't adjacent to the acetoin operon,wasn't regulated by the acetate and quorum sensing.Considering SwrR exerts negative effect on the expression of genes involved in the 2,3-butanediol production,we characterized the ability of swrR mutant to ferment 2,3-butanediol.It showed that the swrR mutant consumed sugar and produced 2,3-butanediol more quickly than the wild strain.3)Cloning and characterization of 2,3-butanediol dehydrogenases in Serratia marcescensThough we have cloned gene slaC which is in charge of converting acetoin to meso-2,3-butanediol,we obtained a second 2,3-butanediol dehydrogenase gene here.The analysis of these two proteins at NCBI revealed that they all belong to short-chain dehydrogenase(SDR)family and there is a close relationship between they and 2,3-butanediol dehydrogenases from K.pneumoniae and B.saccharolyticum.We expressed these two genes in E.coli and purified them.The enzymatic activity showed that SlaC mainly catalyze meso-2,3-butanediol formation from acetoin and SlaCS is mainly in charge of the transformation between acetoin and L-2,3-butanediol.The effects of temperature and pH on the activity of SlaC and SlaCS were investigated by using both ketone reduction and diol oxidation reactions.Maximum activity for diol oxidation of SlaC was observed at 50? and pH7.The optimal temperature and pH for ketone reduction of SlaC were 50? and pH6.Maximum activity for diol oxidation of SlaCS was observed at 60? and pH10.The optimal temperature and pH for ketone reduction of SlaCS were 40? and pH9.The different enzymatic activity between these two enzymes indicated that culture conditions have great effects on the optical purity of 2,3-butanediol produced by Serratia marcescens.All these results provide a basis for the realization of the mechanism of 2,3-butanediol stereoisomer formation and producing optically pure 2,3-butanediol in industry.4)Research on the metabolic engineering of Serratia marcescensWe built an autoinducible expression system in order to metabolic engineer Serratia marcescens.To design a system which is compatible with established protein expression system,we amplified the quorum sensing signal with T7 RNA polymerase and connected the QS controller to the well-known pET-expression vector system..By using this system,we tried to express the slaR gene and two xylose utilization genes in Serratia marcescens,respectively.The results showed the successfully expression of slaR could improve the production of 2,3-butanediol in Serratia marcescens.In the other hand,the genes xylose isomerase and xylulokinase were coexpressed in Serratia marcescens and their products could make Serratia marcescens utilize xylose to produce 2,3-butanediol.All these achievements provide a basis for utilizing lignin-based material as fermentation resources.