Cloning,Expression And Function Studies Of α-Phosphoglucomutase Gene α-PGM2

In the previous study, we isolated an exopolysaccharide (EPS)-producing and intellectual property strain Lactobacillus kefiranofaciens ZW3 from traditional Tibet kefir grain. This strain with good probiotic and fermentation properties also has high capability of EPS production with a maximum amount of 2000 mg/L in the modified MRS medium. The EPS produced by ZW3 shows good structure and physicochemical characteristics. All the results showed that ZW3 is a very promising strain which can be used in food, medicine and other fields.The genome sequence of Lb. kefiranofaciens ZW3 was analyzed in this study, The complete genome of Lb. kefiranofaciens ZW3 contains a single, circular chromosome of 2,113,023 bp and two plasmids (pWW1 [194,769 bp] and pWW2 [46,296 bp]). Moreover, a 14.4 kb EPS gene cluster was also existed in ZW3 genome which contains 17 EPS-related genes and lots of housekeeping genes in the metabolic pathways of exopolysaccharide, such as pgm2,galE,galK.In order to study the Lactobacillus kefiranofaciens ZW3 metabolic pathways, look for the most efficient exopolysaccharide pathways, and build overexpression stains to yield more exopolysaccharide, the pgm2 gene was amplified by the designed primers with ZW3 chromosome as template. Recombinant plasmid pET30a-pgm2 are constructed to transform into E.coli BL21, and expression successfully. Meanwhile, expression vector in lactic acid bacteria was constructed and electroporated into Lactobacillus kefiranofaciens ZW3 to build the overexpression strains. Assessing growth curves found out that they have some differences in overexpression stains with wild bacterias.The phenol-sulfuric acid method was used to study the extracellular polysaccharide production of the recombinant strains with the control of wild bacteria Lactobacillus kefiranofaciens ZW3. And the results showd that extracellular polysaccharide production have a increasing of 20.16%. According to the SDS-PAGE electrophoresis, PGM2 is successfully expressed in Escherichia coli and Lactobacillus kefiranofaciens ZW3. In conclusion, besides extracellular polysaccharide gene cluster, PGM2 also played a very important role in metabolic pathways of extracellular polysaccharide synthesis.