Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel.
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
mBio. 2020 Mar 31;11(2):e00443-20. doi: 10.1128/mBio.00443-20.
is a mesophilic, anaerobic, butanol-producing bacterium, originally isolated from soil. It was recently reported that possesses multiple cellulosomal elements and would potentially form the smallest cellulosome known in nature. Its genome contains only eight dockerin-bearing enzymes, and its unique scaffoldin bears two cohesins (Cohs), three X2 modules, and two carbohydrate-binding modules (CBMs). In this study, all of the cellulosome-related modules were cloned, expressed, and purified. The recombinant cohesins, dockerins, and CBMs were tested for binding activity using enzyme-linked immunosorbent assay (ELISA)-based techniques. All the enzymes were tested for their comparative enzymatic activity on seven different cellulosic and hemicellulosic substrates, thus revealing four cellulases, a xylanase, a mannanase, a xyloglucanase, and a lichenase. All dockerin-containing enzymes interacted similarly with the second cohesin (Coh2) module, whereas Coh1 was more restricted in its interaction pattern. In addition, the polysaccharide-binding properties of the CBMs within the scaffoldin were examined by two complementary assays, affinity electrophoresis and affinity pulldown. The scaffoldin of exhibited high affinity for cellulosic and hemicellulosic substrates, specifically to microcrystalline cellulose and xyloglucan. Evidence that supports substrate-dependent secretion of cellulosomes is presented. The results of our analyses contribute to a better understanding of simple cellulosome systems by identifying the key players in this minimalistic system and the binding pattern of its cohesin-dockerin interaction. The knowledge gained by our study will assist further exploration of similar minimalistic cellulosomes and will contribute to the significance of specific sets of defined cellulosomal enzymes in the degradation of cellulosic biomass. Cellulosome-producing bacteria are considered among the most important bacteria in both mesophilic and thermophilic environments, owing to their capacity to deconstruct recalcitrant plant-derived polysaccharides (and notably cellulose) into soluble saccharides for subsequent processing. In many ecosystems, the cellulosome-producing bacteria are particularly effective "first responders." The massive amounts of sugars produced are potentially amenable in industrial settings to further fermentation by appropriate microbes to biofuels, notably ethanol and butanol. Among the solvent-producing bacteria, has the smallest cellulosome system known thus far. The importance of investigating the building blocks of such a small, multifunctional nanomachine is crucial to understanding the fundamental activities of this efficient enzymatic complex.
是一种嗜温、厌氧、产丁醇的细菌，最初从土壤中分离得到。最近有报道称， 拥有多个纤维小体元件，并且可能形成自然界中已知的最小纤维小体。其基因组仅包含 8 个含有 dockerin 的酶，其独特的支架蛋白含有 2 个粘着(cohesin)（Cohs）、3 个 X2 模块和 2 个碳水化合物结合模块（CBMs）。在这项研究中，所有与纤维小体相关的模块均被克隆、表达和纯化。使用酶联免疫吸附测定（ELISA）技术测试重组粘着(cohesin)、dockerin 和 CBM 的结合活性。所有酶均在七种不同的纤维素和半纤维素底物上进行了比较酶活性测试，从而揭示了四种纤维素酶、木聚糖酶、甘露聚糖酶、木葡聚糖酶和木聚糖内切酶。所有含有 dockerin 的酶与第二个粘着(cohesin)（Coh2）模块的相互作用相似，而 Coh1 的相互作用模式受到更多限制。此外，通过两种互补的测定方法，亲和电泳和亲和下拉，研究了支架蛋白中 CBM 的多糖结合特性。 支架蛋白对纤维素和半纤维素底物表现出高亲和力，特别是对微晶纤维素和木葡聚糖。提出了支持纤维小体依赖底物分泌的证据。我们的分析结果有助于通过鉴定这种简化系统中的关键参与者以及其粘着(cohesin)-dockerin 相互作用的结合模式，更好地理解简单的纤维小体系统。我们研究获得的知识将有助于进一步探索类似的简化纤维小体，并有助于确定纤维素生物质降解中特定的一套定义明确的纤维小体酶的重要性。产纤维小体的细菌在中温和高温环境中被认为是最重要的细菌之一，因为它们能够将植物衍生的抗性多糖（尤其是纤维素）分解成可溶性糖，以便后续加工。在许多生态系统中，产纤维小体的细菌是特别有效的“第一响应者”。产生的大量糖在工业环境中可能适合适当的微生物进一步发酵为生物燃料，特别是乙醇和丁醇。在产溶剂的细菌中， 是迄今为止已知的最小纤维小体系统。研究这种小型多功能纳米机器的构建块对于理解这种高效酶复合物的基本活性至关重要。
