Engineering of the 2,3-butanediol pathway of Paenibacillus polymyxa DSM 365

Christoph Schilling; Rosario Ciccone; Volker Sieber; Jochen Schmid

doi:10.1016/j.ymben.2020.07.009

Engineering of the 2,3-butanediol pathway of Paenibacillus polymyxa DSM 365

Metab Eng. 2020 Sep:61:381-388. doi: 10.1016/j.ymben.2020.07.009. Epub 2020 Aug 7.

Authors

Christoph Schilling¹, Rosario Ciccone¹, Volker Sieber², Jochen Schmid³

Affiliations

¹ Chair of Chemistry of Biogenic Resources, Technical University of Munich, Campus for Biotechnology and Sustainability, Schulgasse 16, 94315, Straubing, Germany.
² Chair of Chemistry of Biogenic Resources, Technical University of Munich, Campus for Biotechnology and Sustainability, Schulgasse 16, 94315, Straubing, Germany; Fraunhofer IGB, Straubing Branch BioCat, Schulgasse 23, 94315, Straubing, Germany; TUM Catalysis Research Center, Ernst-Otto-Fischer-Straße1, 85748, Garching, Germany; The University of Queensland, School of Chemistry and Molecular Biosciences, 68 Copper Road, St. Lucia, 4072, Australia.
³ 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, Norwegian University of Science and Technology, 7034, Trondheim, Norway. Electronic address: jochen.schmid@ntnu.no.

PMID: 32771627
DOI: 10.1016/j.ymben.2020.07.009

Abstract

Paenibacillus polymyxa is a Gram-positive, non-pathogenic soil bacterium that has been extensively investigated for the production of R-,R-2,3-butanediol in exceptionally high enantiomeric purity. Rational metabolic engineering efforts to increase productivity and product titers were restricted due to limited genetic accessibility of the organism up to now. By use of CRISPR-Cas9 mediated genome editing, six metabolic mutant variants were generated and compared in batch fermentations for the first time. Downstream processing was facilitated by completely eliminating exopolysaccharide formation through the combined knockout of the sacB gene and the clu1 region, encoding for the underlying enzymatic machinery of levan and paenan synthesis. Spore formation was inhibited by deletion of spoIIE, thereby disrupting the sporulation cascade of P. polymyxa. Optimization of the carbon flux towards 2,3-butanediol was achieved by deletion of the lactate dehydrogenase ldh1 and decoupling of the butanediol dehydrogenase from its natural regulation via constitutive episomal expression. The improved strain showed 45 % increased productivity, reaching a final concentration of 43.8 g L^-1 butanediol. A yield of 0.43 g g^-1 glucose was achieved, accounting for 86 % of the theoretical maximum.

Keywords: Butanediol; CRISPR-Cas9; Lactate dehydrogenase; Mixed acid pathway; Paenibacillus polymyxa.

MeSH terms

Butylene Glycols / metabolism*
CRISPR-Cas Systems*
Gene Editing*
Metabolic Engineering*
Paenibacillus polymyxa* / genetics
Paenibacillus polymyxa* / metabolism

Substances

Butylene Glycols
2,3-butylene glycol