Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide

Streptococcus thermophilus Sfi6 produces an exopolysaccharide (EPS) compose d of glucose, galactose and N-acetylgalactosamine in the molar ratio of 1:2 :1, The genes responsible for the EPS biosynthesis have been isolated previ ously and found to be clustered in a 14.5 kb region encoding 13 genes. Tran sfer of this gene cluster into a non-EPS-producing heterologous host, Lacto coccus lactis MG1363, yielded an EPS with a similar high molecular weight, but a different structure from the EPS from the native host, The structure of the recombinant EPS was determined by means of H-1 homonuclear and H-1-C -13 heteronuclear two-dimensional nuclear magnetic resonance (NMR) spectra and was found to be --> 3)-beta-D-Glcp-(1 --> 3)-alpha-D-Galp-(1 --> 3)-bet a-D-Galp-(1 --> as opposed to --> 3)[alpha-D-Galp-(1 --> 6)]-D-beta-Glcp-(1 --> 3)-alpha-D-GalpNAc-(1 --> 3)-beta-D-Galp-(1 --> for the wild-type S. t hermophilus Sfi6. Furthermore, L. lactis MG1363 (pFS101) was also lacking a UDP-N-acetylglucosamine C4-epimerase activity, which would provide UDP-Gal NAc for a GalNAc incorporation into the EPS and probably caused the substit ution of GalNAc by Gal in the recombinant EPS, This modification implies th at (i) bacterial glycosyltransferases could potentially have multiple speci ficities for the donor and the acceptor sugar molecule; and (ii) the repeat ing unit polymerase can recognize and polymerize a repeating unit that diff ers in the backbone as well as in the side-chain from its native substrate.