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

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.