Cloning,Expression And Function Studies Of Wang₁297Gene Of Lactobacillus Kefiranofaciens ZW3（CGMCC2809）

Lactic acid bacteria （LAB） are generally recognized as safe food-grade microorganisms, and exopolysaccharide is an important secondary metabolite of LAB. Exopolysaccharide has many functions. The production of exopolysaccharide is relatively low, which has limited industrial applications. How to improve the yield of LAB expolysaccharide is a hot research topic, and understanding the mechanism of expolysaccharide biosynthesis is the key part.The whole genome sequencing of Lactobacillus kefiranofaciens ZW3is already known, it carries a14.4kb EPS gene cluster （WANG₁283-WANG₁299）, which contains17EPS-related genes. These genes showed high similarity to those which are involved in the regulation, polymerization, chain length determination and export of the EPS. In this study, semi-quantitative RT-PCR technique was used to analyze the expression of six time point of EPS genes, WANG₁283、WANG₁285、WANG₁293do not have much change; WANG₁284、WANG₁286、WANG₁287、WANG₁288、 WANG₁290、WANG₁291、WANG₁292、WANG₁294、WANG₁296、WANG₁297、 WANG₁298、WANG₁299have highest expression level at50h、60h, during which EPS was highly synthesized, suggesting that these genes play a role in the polymerization of polysaccharides; WANG₁289has highest expression level at80h, suggesting that it most likely to work in the late stage of EPS biosynthesis; WANG₁295showing a change in expression level.WANG₁297was picked out from EPS genes to be further studied. Bioinformatics analysis showed that it belongs to Fer4_NifH protein superfamily which share a common ATP-binding domain and it has eps_fam multi locus which are related with capsular polysaccharide biosynthesis. The protein is a intracellular protein. Real-time quantitative PCR was done to test the expression level of gene WANG₁297, it has highest expression level at50h、60h, during which EPS was highly synthesized. And also WANG₁297was cloned and expressed in E.coli, showing that it has no kinase activity.Above all the experiments, WANG₁297has no kinase activity and it is most likely to be involved in the formation of a complex to initiate the EPS polymerization.