Artzi Lior, Morag Ely, Barak Yoav, Lamed Raphael, Bayer Edward A
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
Designer Energy Ltd., Rehovot, Israel.
mBio. 2015 May 19;6(3):e00411-15. doi: 10.1128/mBio.00411-15.
Clostridium clariflavum is an anaerobic, cellulosome-forming thermophile, containing in its genome genes for a large number of cellulosomal enzyme and a complex scaffoldin system. Previously, we described the major cohesin-dockerin interactions of the cellulosome components, and on this basis a model of diverse cellulosome assemblies was derived. In this work, we cultivated C. clariflavum on cellobiose-, microcrystalline cellulose-, and switchgrass-containing media and isolated cell-free cellulosome complexes from each culture. Gel filtration separation of the cellulosome samples revealed two major fractions, which were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to identify the key players of the cellulosome assemblies therein. From the 13 scaffoldins present in the C. clariflavum genome, 11 were identified, and a variety of enzymes from different glycoside hydrolase and carbohydrate esterase families were identified, including the glycoside hydrolase families GH48, GH9, GH5, GH30, GH11, and GH10. The expression level of the cellulosomal proteins varied as a function of the carbon source used for cultivation of the bacterium. In addition, the catalytic activity of each cellulosome was examined on different cellulosic substrates, xylan and switchgrass. The cellulosome isolated from the microcrystalline cellulose-containing medium was the most active of all the cellulosomes that were tested. The results suggest that the expression of the cellulosome proteins is regulated by the type of substrate in the growth medium. Moreover, both cell-free and cell-bound cellulosome complexes were produced which together may degrade the substrate in a synergistic manner. These observations are compatible with our previously published model of cellulosome assemblies in this bacterium.
Because the reservoir of unsustainable fossil fuels, such as coal, petroleum, and natural gas, is overutilized and continues to contribute to environmental pollution and CO2 emission, the need for appropriate alternative energy sources becomes more crucial. Bioethanol produced from dedicated crops and cellulosic waste can provide a partial answer, yet a cost-effective production method must be developed. The cellulosome system of the anaerobic thermophile C. clariflavum comprises a large number of cellulolytic and hemicellulolytic enzymes, which self-assemble in a number of different cellulosome architectures for enhanced cellulosic biomass degradation. Identification of the major cellulosomal components expressed during growth of the bacterium and their influence on its catalytic capabilities provide insight into the performance of the remarkable cellulosome of this intriguing bacterium. The findings, together with the thermophilic characteristics of the proteins, render C. clariflavum of great interest for future use in industrial cellulose conversion processes.
明黄梭菌是一种厌氧、形成纤维小体的嗜热菌，其基因组中含有大量编码纤维小体酶的基因以及一个复杂的脚手架蛋白系统。此前，我们描述了纤维小体各组分间主要的黏附素 - dockerin相互作用，并在此基础上推导了多种纤维小体组装模型。在这项研究中，我们在含有纤维二糖、微晶纤维素和柳枝稷的培养基上培养明黄梭菌，并从每种培养物中分离出无细胞纤维小体复合物。对纤维小体样品进行凝胶过滤分离，得到两个主要组分，通过无标记液相色谱 - 串联质谱（LC-MS/MS）对其进行分析，以鉴定其中纤维小体组装的关键成分。在明黄梭菌基因组中存在的13种脚手架蛋白中，鉴定出了11种，并鉴定出了来自不同糖苷水解酶和碳水化合物酯酶家族的多种酶，包括糖苷水解酶家族GH48、GH9、GH5、GH30、GH11和GH10。纤维小体蛋白的表达水平随用于培养该细菌的碳源而变化。此外，还检测了每种纤维小体在不同纤维素底物、木聚糖和柳枝稷上的催化活性。从含微晶纤维素的培养基中分离出的纤维小体是所有测试纤维小体中活性最高的。结果表明，纤维小体蛋白的表达受生长培养基中底物类型的调节。此外，还产生了无细胞和细胞结合的纤维小体复合物，它们可能协同降解底物。这些观察结果与我们之前发表的该细菌纤维小体组装模型一致。
由于煤、石油和天然气等不可持续化石燃料的储量被过度利用，且继续造成环境污染和二氧化碳排放，因此对合适替代能源的需求变得更加迫切。由专用作物和纤维素废料生产的生物乙醇可以提供部分解决方案，但必须开发出一种经济高效的生产方法。厌氧嗜热菌明黄梭菌的纤维小体系统包含大量纤维素分解酶和半纤维素分解酶，这些酶以多种不同的纤维小体结构自组装，以增强对纤维素生物质的降解。鉴定该细菌生长过程中表达的主要纤维小体成分及其对催化能力的影响，有助于深入了解这种有趣细菌非凡纤维小体的性能。这些发现，连同蛋白质的嗜热特性，使得明黄梭菌在未来工业纤维素转化过程中的应用极具吸引力。
