Abstract:
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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. |