Precise promoter integration improves cellulose bioconversion and thermotolerance in Clostridium cellulolyticum.

Lignocellulose has been used for production of sustainable biofuels and value-added chemicals. However, the low-efficiency bioconversion of lignocellulose greatly contributes to a high production cost. Here, we employed CRISPR-Cas9 editing to improve cellulose degradation efficiency by editing a regulatory element of the cip-cel gene cluster in Clostridium cellulolyticum. Insertion of a synthetic promoter (P4) and an endogenous promoter (P2) in the mspI-deficient parental strain (Δ2866) created chromosomal integrants, P4-2866 and P2-2866, respectively. Both engineered strains increased the transcript abundance of downstream polycistronic genes and enhanced in vitro cellulolytic activities of isolated cellulosomes. A high cellulose load of 20 g/L suppressed cellulose degradation in the parental strain in the first 150 h fermentation; whereas P4-2866 and P2-2866 hydrolyzed 29% and 53% of the cellulose, respectively. Both engineered strains also demonstrated a greater growth rate and a higher cell biomass yield. Interestingly, the Δ2866 parental strain demonstrated better thermotolerance than the wildtype strain, and promoter insertion further enhanced thermotolerance. Similar improvements in cell growth and cellulose degradation were reproduced by promoter insertion in the wildtype strain and a lactate production-defective mutant (LM). P2 insertion in LM increased ethanol titer by 65%. Together, the editing of regulatory elements of catabolic gene clusters provides new perspectives on improving cellulose bioconversion in microbes.