Overexpression Of Gene Integrated Into Genome And Expression Analysis Of The Related Promoters In Klebsiella Pneumoniae

As an important industrial strain,Klebsiella pneumoniae has attracted lots of researchers using genetic engineering methods to modify its metabolic pathways,for instance,they tried to weaken or eliminate its byproducts biosynthetic pathway or strengthen the target products synthetic pathway.Therefore,the purpose of this paper is to provide more technic tool and basic data for usage of metabolic engineering.In this study,a new method of gene integrated into genome was established,which is based on the method of Red Recombination.Three-step PCR was designed to construct the target fragment which was consist of four fragments.Using this target fragment,budA+budB(2,3-BD synthesis gene)of genome was successfully replaced by dhaT(including bud promoter),the mutant strain of KG3-2(?lhdA?budA+ budB::Pbud-dhaT)was constructed.After compared the ratio of NADH/NAD+ during the culture,we verified that dhaT was expressed successfully,which was stable and harmless for strain.Due to the integration into genome,it became single-copy and produced less protein.So,its ability to improve intracellular redox balance was weaker than that of KG3-1(contained the vector of pETbud::dhaT),which could be solved by using a strong promoter for integrated-gene over-expressed.Therefore,in this study we used the lacZ reporter gene to compare the actual expression level of promoters(Pbud,PT5 and Ptac)on different carbon sources in K.pneumoniae systematically.The results indicated that promoters PT5 and Ptac,designed for Escherichia coli,were functional in K.pneumoniae.The basal expression level of Ptac reached to 396.5 U/mg,which was 9.5-fold higher when compared with PT5 in LB medium,indicating Ptac could be used as an efficient "constitutive" promoter as well as an efficient induced promoter in K.pneumonia.In different carbon sources medium,the newly constructed constitutive endogenous Pbud proved to be a stable and weak promoter.On the basis of our data,a set of Pbud and Ptac promoters could meet the broad range(about 1000 orders of magnitude)of gene expression needs for engineering K.pneumoniae.