Structural elucidation of the fucose containing polysaccharide of Paenibacillus polymyxa DSM 365

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

doi:10.1016/j.carbpol.2021.118951

Structural elucidation of the fucose containing polysaccharide of Paenibacillus polymyxa DSM 365

Carbohydr Polym. 2022 Feb 15:278:118951. doi: 10.1016/j.carbpol.2021.118951. Epub 2021 Dec 2.

Authors

Christoph Schilling¹, Leesa J Klau², Finn L Aachmann³, Broder Rühmann⁴, Jochen Schmid⁵, Volker Sieber⁶

Affiliations

¹ Chair of Chemistry of Biogenic Resources, Technical University of Munich, Campus for Biotechnology and Sustainability, Schulgasse 16, 94315 Straubing, Germany. Electronic address: christoph.schilling@tum.de.
² Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway. Electronic address: leesa.klau@ntnu.no.
³ Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway. Electronic address: finn.l.aachmann@ntnu.no.
⁴ Chair of Chemistry of Biogenic Resources, Technical University of Munich, Campus for Biotechnology and Sustainability, Schulgasse 16, 94315 Straubing, Germany. Electronic address: broder.ruehmann@tum.de.
⁵ Chair of Chemistry of Biogenic Resources, Technical University of Munich, Campus for Biotechnology and Sustainability, Schulgasse 16, 94315 Straubing, Germany; Department of Biotechnology and Food Science, NTNU Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway; Institute for Molecular Microbiology and Biotechnology, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany. Electronic address: jochen.schmid@uni-muenster.de.
⁶ Chair of Chemistry of Biogenic Resources, Technical University of Munich, Campus for Biotechnology and Sustainability, Schulgasse 16, 94315 Straubing, Germany; School of Chemistry and Molecular Biosciences, The University of Queensland, 68 Copper Road, St. Lucia 4072, Australia; TUM Catalysis Research Center, Ernst-Otto-Fischer-Straße1, 85748, Garching, Germany. Electronic address: sieber@tum.de.

PMID: 34973768
DOI: 10.1016/j.carbpol.2021.118951

Abstract

Paenibacillus polymyxa is an avid producer of exopolysaccharides of industrial interest. However, due to the complexity of the polymer composition, structural elucidation of the polysaccharide remained unfeasible for a long time. By using a CRISPR-Cas9 mediated knock-out strategy, all single glycosyltransferases as well as the Wzy polymerases were individually deleted in the corresponding gene cluster for the first time. Thereby, it was observed that the main polymer fraction was completely suppressed (or deleted) and a pure minor fucose containing polysaccharide could be isolated, which was named paenan II. Applying this combinatorial approach, the monosaccharide composition, sequence and linkage pattern of this novel polymer was determined via HPLC-MS, GC-MS and NMR. Furthermore, we demonstrated that the knock-out of the glycosyltransferases PepQ, PepT, PepU and PepV as well as of the Wzy polymerase PepG led to the absence of paenan II, attributing those enzymes to the assembly of the repeating unit.

Keywords: CRISPR-Cas9; Exopolysaccharides; Fucose; Paenan; Paenibacillus polymyxa.

MeSH terms

Carbohydrate Conformation
Fucose / chemistry*
Paenibacillus polymyxa / chemistry*
Polysaccharides, Bacterial / chemistry*

Substances

Polysaccharides, Bacterial
Fucose