Tao Xuanyu, Xu Tao, Kempher Megan L, Liu Jiantao, Zhou Jizhong
Institute for Environmental Genomics, Department of Microbiology and Plant Biology, and School of Civil Engineering and Environmental Sciences, University of Oklahoma, Norman, OK, USA.
Institute for Environmental Genomics, Department of Microbiology and Plant Biology, and School of Civil Engineering and Environmental Sciences, University of Oklahoma, Norman, OK, USA; Section on Pathophysiology and Molecular Pharmacology, Joslin Diabetes Center, Boston, MA, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA.
Metab Eng. 2020 Jul;60:110-118. doi: 10.1016/j.ymben.2020.03.013. Epub 2020 Apr 12.
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生物燃料和高附加值化学品。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。在此，我们利用CRISPR-Cas9编辑技术，通过编辑解纤维梭菌中cip-cel基因簇的一个调控元件来提高纤维素降解效率。在缺乏mspI的亲本菌株（Δ2866）中分别插入一个合成启动子（P4）和一个内源启动子（P2），创建了染色体整合体，分别为P4-2866和P2-2866。这两种工程菌株均增加了下游多顺反子基因的转录丰度，并增强了分离出的纤维小体的体外纤维素分解活性。在150小时的发酵过程中，20 g/L的高纤维素负荷抑制了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。这两种工程菌株还表现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。在野生型菌株和乳酸生产缺陷型突变体（LM）中通过启动子插入也再现了细胞生长和纤维素降解方面的类似改善。在LM中插入P2使乙醇滴度提高了65%。总之，分解代谢基因簇调控元件的编辑为改善微生物中的纤维素生物转化提供了新的视角。
