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

Christoph Schilling, Leesa J. Klau, Finn L. Aachmann, Broder Rühmann, Jochen Schmid, Volker Sieber

Abstract

Paenibacillus polymyxa is a Gram-positive soil bacterium known for producing a wide range of exopolysaccharides. 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-β-D-Glc, 14-β- Man and a 1,3,4-branching β-D-Gal residue with a sidechain comprising of a terminal β-D-Gal3,4-Pyr and 13-β-D-Glc. For paenan III, results indicated a backbone consistingof 13-β-D-Glc, 1,3,4-linked α-D-Man and 1,3,4-linked α-D-GlcA. NMR analysis indicated monomeric β-D-Glc and α-D-Man sidechains for the branching Man and GlcA residues respectively.