Using gene editing to investigate the function of epsD gene in exopolysaccharide (EPS) biosynthesis of Lactococcus lactis subsp. cremoris JFR1

Abstract

Lactococcus lactis JFR1, a ropy and EPS producing strain, is used to modify texture in dairy products. The mechanism of EPS biosynthesis in JFR1 is still not completely understood yet. Multiple alignment of the epsD gene of JFR1, which is the homolog of the epsE gene (the priming glycosyltranferase) of Streptococcus thermophilus, showed tyrosine residues conserved at Y161, Y198, and Y199. Direct editing of the rpoB gene of JFR1 was carried out as a control to generate a rifampicin-resistant JFR1 strain using ssDNA recombineering with a constructed strain (JPP). Only the point mutation of the rpoB gene produced the rifampincin-resistant phenotype. JPP was shown to function for mutagenesis with Lactococcus lactis strain JFR1 and was then used to directly edit the target gene epsD. Tyrosine residues either at Y198 or Y199 could act as phosphorylation sites of EpsD to activate the phosphoglycosyltransferase during synthesis of EPS in JFR1. EPS production of two mutant strains (Y198 and Y199) was lower than the EPS levels produced by wild type strain JFR1. Moreover, these strains, which were both from mutant tyrosine residues at Y198 and Y199 of the epsD gene in JFR1 were non-ropy, but wild-type of Lactococcus lactis JFR1 displayed a ropy status. It demonstrated that tyrosine mutation of the epsD gene could impact EPS biosynthesis in JFR1.