The Function Of BudC Gene Of Bacillus Licheniformis And Construction Of Strains For Enhanced Production Of Acetoin

Acetoin has broad applications in fields of food,pharmaceutical,tobacco,chemical industry etc.Microorganisms can produce acetoin.As generally recognized as safe strain,Bacillus licheniformis can get high yield of acetoin and meet applications in fields of food,pharmaceutical and tobacco.B.licheniformis can catalyze reversible reaction between acetoin and 2,3-butanediol.To date,the gene encoding 2,3-butanediol dehydrogenase(also known as acetoin reductase),responsibe for this reversible reaction,has not yet been confirmed experimentally.Gene budC from the genome database of B.licheniformis is annotated as 2,3-butanediol dehydrogenase or acetoin reductase,but its specific function remains unclear so far,lack of experimental verification.The objective of this study was to explore the specific function of budC gene of B.licheniformis and the relationship between budC and acetoin metabolism;and as a basis,construct genetically engineered strains for enhanced production of acetoin.By means of homology sequence search and analysis,another member of our research group found that yjmD gene might encode 2,3-butanediol dehydrogenase.In this study,by means of gene knockout method,we constructed the yjmD knockout strain B.licheniformis WX-02 ?yjmD.For acetoin fermentation,compared to the wild strain,the loss of yjmD did not have an impact on the production of acetoin,configuration and production of 2,3-butanediol.Therefore,the yjmD gene of B.licheniformis had no direct relationship with acetoin metabolism.By means of gene knockout,budC deletion strain was constructed,designated B.licheniformis WX-02 ?budC.In early and medium fermentation stage this strain abolished meso-2,3-butanediol production and accumulated the precursor acetoin.At the same time,no meso-2,3-butanediol dehydrogenase activity was detected;acetoin reductase activity decreased and D-(-)-2,3-butanediol dehydrogenase activity was not affected in the cell extract of the budC deletion strain.Complementation of the budC knockout strain was performed by introducing a budC expression plasmid and generated B.licheniformis WX-02 ?budC/pHYbudC.This strain could restore characteristics of the wild strain,producing meso-2,3-butanediol and having meso-2,3-butanediol dehydrogenase activity in early and medium fermentation stage.In addition,B.licheniformis WX-02 ?budC produced a small amount of meso-2,3-butanediol and had weak meso-2,3-butanediol dehydrogenase activity in late fermentation stage.From the results above,we could conclude that budC of B.licheniformis encoded the main meso-2,3-butanediol dehydrogenase,catalyzing the reversible reaction between acetoin and meso-2,3-butanediol,and wasn't responsible for the conversion between acetoin and D-(-)-2,3-butanediol.This was the first research about the function of budC gene from B.licheniformis.We investigated the acetoin fermentation process of B.licheniformis WX-02 ?budC.The deletion of budC partly blocked the conversion from acetoin to 2,3-butanediol(meso-2,3-butanediol).As a result,the production of byproduct 2,3-butanediol decreased and the acetoin production was improved.The highest production of acetoin was achieved at 39.83 g/L,27.05%higher than that of the wild strain B.licheniformis WX-02.Deletion of ldh,lactate dehydrogenase gene annotated from NCBI genome database and KEGG pathway database,was conducted on the basis of B.licheniformis WX-02?budC,generating the strain B.licheniformis WX-02 ?budC ?ldh.For acetoin fermentation,this strain didn't decrease the production of byproduct lactate,but increased the production of lactate and acetate and decreased acetoin production.On the basis of B.licheniformis WX-02 ?budC,we deleted acoR gene,the positive regulator gene of acetoin dehydrogenase system,responsible acetoin catabolism,generating the strain B.licheniformis WX-02 ?budC ?acoR.The strain could,to some extent,weaken the catabolism of acetoin.The acetoin production of B.licheniformis WX-02 ?budC ?acoR reached 44.32 g/L,11.27%higher than that of B.licheniformis WX-02 ?budC,41.37%higher than that of B.licheniformis WX-02.The apparent efficiency of glucose conversion of this strain reached 44.32%.Genetically engineered strain B.licheniformis WX-02 ?budC ?acoR could enhance acetoin production.