Abstract:

In the phosphotransferase system of Bacillus amyloliquefaciens,glucose is transported into the cell mainly through enzymes EI,HPr and EII^Glc encoded by the ptsGHI operon. In this investigation,first,about 1 kbp upstream and downstream DNA fragments of ptsG and ptsHI genes were amplified and were ligated into one fragment by using fusion PCR. Then,the fused fragment was inserted into the temperature-sensitive plasmid pKS2 and transformed by electroporation into Bacillus amyloliquefaciens XH7,thus constructing ptsG- and ptsHI-deficient strains. Experimental results show that ( 1) in LB medium,there is no obvious difference in the growth characteristics between the deficient strains and the wild-type strains; ( 2) in LB medium supplemented with 2% glucose,the ptsG gene-deficient strain shows a maximal cell density that is 28% higher than the wild-type one as well as a longer steady period than the control,while the ptsHI gene-deficient strain shows a maximal cell density that is about 32% lower than the wild-type one,and its growth accords well with that in LB medium; and ( 3) in guanosine fermentation experiments,the guanosine production yield of the ptsG gene-deficient strain is about 24% higher than that of the wild-type one,while that of the ptsHI gene-deficient strain is about 82% lower These results demonstrate that ptsG gene-deficient strains are of good ability in growth and guanosine biosynthesis.

Key words: Bacillus amyloliquefaciens, phosphotransferase system, gene knockout, metabolic engineering