Hong Wei, Zhang Jie, Feng Yingang, Mohr Georg, Lambowitz Alan M, Cui Gu-Zhen, Liu Ya-Jun, Cui Qiu
Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, P R China ; University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, P R China.
Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, P R China.
Biotechnol Biofuels. 2014 May 29;7:80. doi: 10.1186/1754-6834-7-80. eCollection 2014.
Clostridium thermocellum is a thermophilic anaerobic bacterium that degrades cellulose by using a highly effective cellulosome, a macromolecular complex consisting of multiple cellulose degrading enzymes organized and attached to the cell surface by non-catalytic scaffoldins. However, due largely to lack of efficient methods for genetic manipulation of C. thermocellum, it is still unclear how the different scaffoldins and their functional modules contribute to cellulose hydrolysis.
We constructed C. thermocellum mutants with the primary scaffoldin CipA (cellulosome-integrating protein A) truncated at different positions or lacking four different secondary scaffoldins by using a newly developed thermotargetron system, and we analyzed cellulose hydrolysis, cellulosome formation, and cellulose binding of the mutants. A CipA truncation that deletes six type I cohesin modules, which bind cellulolytic enzymes, decreased cellulose hydrolysis rates by 46%, and slightly longer truncations that also delete the carbohydrate binding module decreased rates by 89 to 92%, indicating strong cellulosome-substrate synergy. By contrast, a small CipA truncation that deletes only the C-terminal type II dockerin (XDocII) module detached cellulosomes from the cells, but decreased cellulose hydrolysis rates by only 9%, suggesting a relatively small contribution of cellulosome-cell synergy. Disruptants lacking any of four different secondary scaffoldins (OlpB, 7CohII, Orf2p, or SdbA) showed moderately decreased cellulose hydrolysis rates, suggesting additive contributions. Surprisingly, the CipA-ΔXDocII mutant, which lacks cell-associated polycellulosomes, adheres to cellulose almost as strongly as wild-type cells, revealing an alternate, previously unknown cellulose-binding mechanism.
Our results emphasize the important role of cellulosome-substrate synergy in cellulose degradation, demonstrate a contribution of secondary scaffoldins, and suggest a previously unknown, non-cellulosomal system for binding insoluble cellulose. Our findings provide new insights into cellulosome function and impact genetic engineering of microorganisms to enhance bioconversions of cellulose substrates.
嗜热栖热梭菌是一种嗜热厌氧菌，它通过使用一种高效的纤维小体来降解纤维素，纤维小体是一种大分子复合物，由多种纤维素降解酶组成，并通过非催化性支架蛋白组织并附着在细胞表面。然而，很大程度上由于缺乏对嗜热栖热梭菌进行基因操作的有效方法，目前仍不清楚不同的支架蛋白及其功能模块如何促进纤维素水解。
我们使用新开发的热靶向基因敲除系统构建了在不同位置截短主要支架蛋白CipA（纤维小体整合蛋白A）或缺失四种不同次要支架蛋白的嗜热栖热梭菌突变体，并分析了这些突变体的纤维素水解、纤维小体形成和纤维素结合情况。截短删除六个结合纤维素分解酶的I型黏连蛋白模块的CipA，使纤维素水解速率降低了46%，而截短长度稍长且还删除了碳水化合物结合模块的情况使水解速率降低了89%至92%，这表明纤维小体 - 底物之间存在很强的协同作用。相比之下，仅删除C末端II型dockerin（XDocII）模块的小截短CipA使纤维小体从细胞上脱离，但纤维素水解速率仅降低了9%，这表明纤维小体 - 细胞协同作用的贡献相对较小。缺失四种不同次要支架蛋白（OlpB、7CohII、Orf2p或SdbA）中任何一种的缺失突变体显示纤维素水解速率适度降低，表明存在累加作用。令人惊讶的是，缺乏与细胞相关的多纤维小体的CipA - ΔXDocII突变体与野生型细胞一样强烈地附着在纤维素上，揭示了一种以前未知的交替纤维素结合机制。
我们的结果强调了纤维小体 - 底物协同作用在纤维素降解中的重要作用，证明了次要支架蛋白的贡献，并提出了一种以前未知的非纤维小体系统用于结合不溶性纤维素。我们的发现为纤维小体功能提供了新的见解，并影响微生物基因工程以增强纤维素底物的生物转化。
