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.

木质纤维素已被用于生产可持续的生物燃料和增值化学品。然而，木质纤维素的低效率生物转化极大地导致了高生产成本。在这里，我们采用CRISPR-Cas9编辑来通过编辑解纤梭菌cip-cel基因簇的调控元件来提高纤维素降解效率。在缺乏mspI的亲本菌株（Δ2866）中插入合成启动子（P4）和内源启动子（P2）分别产生了染色体整合体P4-2866和P2-2866。两种工程菌株均增加了下游多顺反子基因的转录本丰度并增强了体外分离的纤维素体的纤维素分解活性。20 g / L的高纤维素负荷抑制了最初150 h发酵中亲本菌株中的纤维素降解；而P4-2866和P2-2866分别水解了29％和53％的纤维素。两种工程菌株还显示出更高的生长速率和更高的细胞生物量产量。有趣的是，Δ2866亲本菌株表现出比野生型菌株更好的耐热性，启动子插入进一步增强了耐热性。通过将启动子插入野生型菌株和乳酸生产缺陷型突变体（LM），可以再现细胞生长和纤维素降解的类似改善。LM中P2的插入使乙醇效价提高了65％。一起，