Site-directed mutagenesis of the glucansucrase gtf180 gene from Lactobacillus reuteri strain 180 was used to transform the active site region. The R-D-glucan (mEPS-PNNS) produced by the triple mutant V1027P:S1137N: A1139S differed in structure from that of the wild-type R-D-glucan (EPS180). Besides (R1f3) and (R1f6) linkages, as present in EPS180, mEPS-PNNS also contained (R1f4) linkages. Linkage analysis, periodate oxidation, and 1D/2D 1 H NMR spectroscopy of the intact mEPS-PNNS, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of mEPS-PNNS afforded a composite model, which includes all identified structural features.