Genetic Function Of Encoding Dhak In Klebsiella Pneumoniae

Klebsiella pneumoniae is used for biological production of1,3-propanediol. The metabolic pathway from glycerol to1,3-propanediol is consists of4key enzymes encoded by the dha operon. Dihydroxyacetone Kinase (DHAK), is one of key enzymes, catalyzes the conversion of the dihydroxyacetone to dihydroxyacetonephosphate. The DHAK contains DHAKⅠ and DHAKⅡ, DHAKⅠ is encoded by dhak, but DHAKⅡ is encoded by dhak1,dhak2and dhak3. The function of genes encoding DHAK in the dha operon was studied in this paper. Firstly, a homologous recombination system applicable to K. pneumoniae TUAC01was constructed. Using this system,4ORFs related to DHAK were selectively knocked-out. The activities of3enzymes in dha operon were assayed in both culture of the mutant strains. The results suggest that DHAKⅡ was a regulatory protein in the dha operon,and the deletion of dhak1and dhak2resulted in the loss of the dha operon expression.In the section of gene cloning, according to the published genome sequence of K. pneumoniae MGH78578, genes encoding DHAK in K. pneumoniae TUAC01were successfully amplified by PCR. The sequences encoding DHAKⅠ and DHAKⅡ were confirmed by homology analysis.In the section of optimization of electroporation parameters, the parameters including pulser voltage, cell growth stage, final electro-competent cell density, EDTA level in medium and plasmid concentration were investigated in detail. It was shown that adding EDTA to a final concentration of0.7mmol/L in the medium, collecting cell at OD6000.7, concentrating the cells to a final density of larger than OD60030, selecteing pulser voltage of2KV with2mm electroporation chamber, the electroporation efficiency reached8.58×105transformants/μg DNA.In the section of gene homologous recombination system construction, plasmid pDK6-red expressing the Red recombinase and plasmid pDK6-flp expressing the FLP recombinase were constructed based on plasmids of pIJ790, pCP20and pDK6. The protocol of gene knockout using the Red recombinase mediated homologous recombination system was constructed in K. pneumoniae TUAC01.With the help of the Red recombinase system,4ORFs of genes encoding DHAK were selectively deleted in K. pneumoniae TUAC01, then6mutant strains were obtained, named as kp△dhak, kp△dhak1,kp△dhak2, kp△dhak3, kp△dhak12and kp△DHAK, respectively.1,3-propanediol fermentation experiments and Enzyme assay showed that the strains lacking the ATP dependent DHAKI gene dhak had no obvious effect on1,3-propanediol production compared with the wild type strain. It also implied that the phosphoenolpyruvate dependent DHAKII was a regulator protein in dha operon, and the dhak1and dhak2genes are necessary for the expression of the dha operon.