CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, PR China; Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, PR China; Shandong Engineering Laboratory for Single Cell Oil, Qingdao Engineering Laboratory for Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, PR China; Dalian National Laboratory for Clean Energy, Qingdao 266101, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel; Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 8499000, Israel.
Bioresour Technol. 2021 Oct;337:125441. doi: 10.1016/j.biortech.2021.125441. Epub 2021 Jun 23.
Consolidated bio-saccharification (CBS) technology employs cellulosome-producing bacterial cells, rather than fungal cellulases, as biocatalysts for cost-effective production of lignocellulosic sugars. Extracellular β-glucosidase (BGL) expression in the whole-cell arsenal is indispensable, due to severe cellobiose inhibition of the cellulosome. However, high-level BGL expression in Clostridium thermocellum is challenging, and the optimal BGL production level for efficient cellulose saccharification is currently unknown. Herein, we obtained new CBS biocatalysts by transforming BGL-expressing plasmids into C. thermocellum, which produced abundant BGL proteins and hydrolyzed cellulose effectively. The optimal ratio of extracellular BGL-to-cellulosome activity was determined to be in a range of 5.5 to 21.6. Despite the critical impact of BGL, both excessive BGL expression and its assembly on the cellulosome via type I cohesin-dockerin interaction led to reduced cellulosomal activity, which further confirmed the importance of coordinated BGL expression with the cellulosome. This study will further promote industrial CBS application in lignocellulose conversion.
整合生物糖化（CBS）技术采用产细胞外纤维二糖酶的细菌细胞，而不是真菌纤维素酶，作为生物催化剂，以经济高效地生产木质纤维素糖。由于细胞外β-葡萄糖苷酶（BGL）对纤维二糖酶的严重抑制，整个细胞武器库中的细胞外 BGL 表达是必不可少的。然而，在高温产纤维素梭菌中高水平表达 BGL 具有挑战性，并且目前尚不清楚有效纤维素糖化的最佳 BGL 生产水平。在此，我们通过将表达 BGL 的质粒转化为高温产纤维素梭菌，获得了新的 CBS 生物催化剂，该催化剂产生了丰富的 BGL 蛋白并有效地水解纤维素。确定最佳的细胞外 BGL-纤维素酶活性比在 5.5 到 21.6 之间。尽管 BGL 具有重要影响，但过多的 BGL 表达及其通过 I 型粘着蛋白-附着蛋白相互作用在细胞外纤维二糖酶上的组装都导致了细胞外纤维二糖酶活性降低，这进一步证实了与细胞外纤维二糖酶协调表达 BGL 的重要性。本研究将进一步推动木质纤维素转化中 CBS 的工业应用。
