CRISPR-Cas9 driven structural elucidation of the heteroexopolysaccharides from Paenibacillus polymyxa DSM 365

Paenibacillus polymyxa is a Gram-positive soil bacterium known for producing a wide range of exopoly-saccharides. However, due to the biopolymer's complexity, structural elucidation has so far been inconclusive. Combinatorial knock-outs of glycosyltransferases were generated in order to separate distinct polysaccharides produced by P. polymyxa. Using a complementary analytical approach consisting of carbohydrate fingerprints, sequence analysis, methylation analysis as well as NMR spectroscopy, the structure of the repeating units of two additional heteroexopolysaccharides termed paenan I and paenan III were elucidated. Results for paenan I identified a trisaccharide backbone consisting of 1-4-beta-D-Glc, 1-4-beta-D-Man and a 1,3,4-branching beta-D-Gal res-idue with a sidechain comprising of a terminal beta-D-Gal3,4-Pyr and 1-3-beta-D-Glc. For paenan III, results indicated a backbone consisting of 1-3-beta-D-Glc, 1,3,4-linked alpha-D-Man and 1,3,4-linked alpha-D-GlcA. NMR analysis indicated monomeric beta-D-Glc and alpha-D-Man sidechains for the branching Man and GlcA residues respectively.

Automated summary

Combinatorial knock-outs of glycosyltransferases were used to identify the repeating units of two new heteroexopolysaccharides, paenan I and paenan III, produced by P. polymyxa.