CRISPR/Cas9-directed engineering of Clostridium beijerinckii enhanced butanol production from whey permeate

Whey permeate (WP), an abundant, lactose-rich byproduct of the dairy industry, constitutes an environmental and economic burden on the industry. Despite the high fermentable sugar content of WP, its bioconversion into biobutanol is limited by the poor lactose-utilization culminating in low solvent productivity by native solventogenic microorganisms. In this study, we report CRISPR/Cas9-mediated genome engineering of Clostridium beijerinckii NCIMB 8052 (hereafter, C. beijerinckii) to improve butanol production from lactose-rich WP. A native putative lactose transporter encoded by Cbei_4545 and an aldehyde reductase (mgR) from Clostridium pasteurianum were integrated into the chromosome of C. beijerinckii at the acetoacetate decarboxylase gene locus to improve lactose utilization and solvent production. The resulting recombinant strain was evaluated alongside the wildtype using lactose, untreated WP, and ion exchange (Amberlite™) resin-treated WP. Across all feedstocks tested, the recombinant strain consistently outperformed the wildtype, producing peak butanol titers of 7.9 g/L, 9.7 g/L, and 20.9 g/L on lactose, WP, and resin-treated WP, respectively, compared to 4.3 g/L, 7.1 g/L, and 11.3 g/L for the wildtype. These results demonstrate the utility of targeted CRISPR/Cas9 genome engineering to substantially improve lactose utilization and butanol production in C. beijerinckii. In this work we constructed a strain of C. beijerinckii with strong potential for conversion of abundant and cost-effective WP to biobutanol.