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.

多粘类芽孢杆菌是一种革兰氏阳性、非致病性土壤细菌，已被广泛研究用于以极高的对映体纯度生产R-,R -2,3-丁二醇。由于迄今为止生物体的遗传可及性有限，提高生产力和产品效价的合理代谢工程努力受到限制。通过使用 CRISPR-Cas9 介导的基因组编辑，首次在批量发酵中生成和比较了六种代谢突变体。通过联合敲除sacB基因和clu1基因完全消除胞外多糖的形成，促进了下游加工区域，编码levan和paenan合成的潜在酶机制。spoIIE的缺失抑制了孢子的形成，从而破坏了多粘假单胞菌的孢子形成级联。通过乳酸脱氢酶ldh1的缺失和丁二醇脱氢酶通过组成型附加体表达将丁二醇脱氢酶从其自然调节中解耦来优化朝向 2,3-丁二醇的碳通量。改进后的菌株生产力提高了 45%，最终浓度达到 43.8 g L ^-1丁二醇。实现了 0.43 g g ^-1葡萄糖的产量，占理论最大值的 86%。