Bud Gene Overexpression Klebsiella Oxytoca HD79 High-yield 2,3-butanediol Engineering Strain Construction

2,3-butanediol(2,3-BD)is a kind of extremely valuable chemical material and novel fuel,which can be widely used in chemical,food,medicine,aerospace and many other fields.2,3-BD can be produced by chemical method and biological method.However,the costly,complicated and pollutional way of chemical synthesis leads to strong interests in microbial production of 2,3-BD.While the main problem of large-scale production of 2,3-BD by microbial method at present is the lower yield of 2,3-BD.Thus attempts have been taken to enhance the productivity of 2,3-BD such as the optimization of fermentation conditions,the mutation breeding and metabolic regulation and control of strains,however,unsatisfactory results has been acquired.With the application of metabolic network theory,3 key enzymes have been found with significant roles in 2,3-BD synthesis pathway of the central carbon metabolism in Klebsiella oxytoca,which are Acetolactate decarboxylase(budA),Acetolactate synthase(budB)and 2,3-BD dehydrogenase(budC).Theoretically,2,3-BD production can be remarkably enhanced by over-expression of the three key enzymes that results in more flux of carbon source to 2,3-butanediol metabolic pathway.In this paper,construction of engineering strains with the Klebsiella oxytoca HD79 as the original strain had been built.The engineering strains were constructed with different over-expressions of BudA,BudB and BudC and the 2,3-BD production were determined.Expression plasmid of pRSFDuet-1 was applied to construct the recombinant plasmids with BudA,BudB and BudC respectively.The recombinant vectors were translated into K.oxytoca HD79 by electric conversion and seven engineering strains named HD79-1,HD79-2,HD79-3,HD79-4,HD79-5,HD79-6,HD79-7 respectively were obtained.Compared with K.oxytoca HD79,fermentation experiments in shaking flasks of the seven engineering strains were performed with glucose as the only carbon source.The glucose consumption and 2,3-BD production were detected by HPLC.According to the results,capacity of engineering strains were higher than that of K.oxytoca HD79,while the highest 2,3-BD yield of the seven strains was obtained in HD79-4 strain that was33.357 g/L,34.80% higher than the original strain;similarly,the conversion rate production strength of 2,3-BD of the HD79-4 strain were 23.68 % and 56.95% higher than that of Klebsiella oxytoca HD79 respectively.Furthermore,mRNA expressions of BudA and BudB in HD79-4 strain were detected by qRT-PCR,which were 39.43 times and 51.25 times more than that of K.oxytoca HD79 respectively.Meanwhile,protein expression differences of target genes in both the engineering strains and the original strain were detected by SDS-PAGE,the results showed that distinct protein bands appeared at 60 kDa and 29 kDa,and the expression levels of the two proteins were higher than the original strain;enzyme activitives of acetolactate synthase and acetolactate decarboxylase were significantly higher than the original strain,increased by 4.35 times and 3.24 times respectively.The study will provide some referential value to learn more about the connections between the expression levels of the three key enzymes in 2,3-BD synthesis route and the yield of 2,3-BD as well as lay the foundation of the industrialization of 2,3-BD production in some degree.