Analysis Of Genome Sequence Of Lactobacillus Kefiranofaciens And Study On Wang₁296Gene

In the previous study, we isolated an exopolysaccharide （EPS）-producing strain Lactobacillus kefiranofaciens ZW3from traditional Tibet kefir grain. This strain has good probiotic and fermentation properties, ZW3also has high capability of EPS production with a maximum amount of1600mg/L. The EPS produced by ZW3shows good structure and physicochemical characteristics. All results showed that ZW3is a very promising strain which can be used in a variety of fields.The genome sequence of Lb. kefiranofaciens ZW3was analyzed in this study, The complete genome of Lb. kefiranofaciens ZW3contains a single, circular chromosome of2,113,023bp and two plasmids （pWWl [194,769bp] and pWW2[46,296bp]）. The overall GC content of the chromosome is37.70%whereas the two plasmids have GC contents of33.98%and36.02%, respectively. The chromosome contains1,908predicted protein-encoding sequences （CDSs）. Plasmids pWW1and pWW2carry199and55predicted protein-encoding sequences, respectively. A14.4kb EPS gene cluster was also existed in ZW3genome which contains17EPS-related genes. Homology analysis of these17genes showed that, there are eight transferase genes, three regulator genes, one chain length determinant gene, two transferation gene, and five unknown genes.We amplified the genes namely WANG₁283-WANG₁299related to EPS biosynthesis with ZW3chromosome as template. Eight of the genes were inserted into plasmid pMG36e for the recombinant expression. The recombinant expression vectors were electroporated into Lactococcus lactis WH-C1. Assessing even growth curves found out that they have some differences.WANG₁296was choosen for further studies. Comparative analysis showed that WANG₁296gene encodes a tyrosine protein phosphatase. Bioinformatics analysis of WANG1296gene showed that this gene contains one open reading frame （ORF） and encodes a protein composed of256amino acids. WANG1296protein is a hydrophobic protein. Transmembrane helices and signal peptide were not defected so that it belongs to the non-secretory intracellular protein. SDS-PAGE analysis showed that WANG1296protein was successfully expressed in Lactococcus lactis WH-C1. The exopolysaccharide yield of the recombinant was1.8times higher than that of the wild stain as analyzed by phenol-sulfuric acid method. The exopolysaccharide yield was significantly improved. According to our experiment and the references, it was concluded WANG1296plays an up regulated role in the biosynthesis of EPS. This is consistent with the sequencing result that WANG₁296is a gene related to EPS biosynthesis.