Abstract: In order to explore the reason for reduced exopolysaccharide (EPS) production by a mutant strain 2# compared with Lactobacillus kefiranofaciens ZW3, we conducted comparative genome-wide analysis of the wild and mutant strains by bioinformatics methods. We also analyzed the role of the mutant genes in metabolic pathways. To evaluate their correlation with EPS production from lactic acid bacteria, we analyzed the relative expression levels of the mutant genes that may be associated with polysaccharide production using RT-qPCR, and constructed a recombinant plasmid carrying the genes with significantly different expression levels and transformed it into Escherichia coli, and then determined the expressed serine/threonine protein kinases (STPKs) activity. The whole genome analysis showed that there were 60 genes mutated in the CDS or the promoter regions, and at least six of them were associated with catalytic domains, namely WANG_0173, WANG_0174, WANG_0175 (encoding dihydroxyacetone kinase or its subunit), WANG_1108 (serine threonine protein kinase), WANG_0292 (synonymous mutation, beta-galactosidase small subunit), and WANG_0840 (udp-n-acetylmuramoylalanine-d-glutamate ligase, MurD). The results showed that the relative expression of WANG_1108 was significantly down-regulated as revealed by RT-qPCR. Therefore, we chose this gene as the target gene related to EPS synthesis to further study the molecular mechanism. It was expressed in E. coil BL21 (DE3) with plasmid pET28a as plasmid vector to identify its function. The expressed product was then detected by SDS-PAGE and the enzyme activity was measured. It was found that the mutant STPKs activity was 37% lower than that of the wild strain, indicating that the mutation of WANG_1108 could partially account for decreased EPS production.

Key words: Lactobacillus kefiranofaciens ZW3, mutant 2#, exopolysaccharide