Brunecky Roman, Chung Daehwan, Sarai Nicholas S, Hengge Neal, Russell Jordan F, Young Jenna, Mittal Ashutosh, Pason Patthra, Vander Wall Todd, Michener William, Shollenberger Todd, Westpheling Janet, Himmel Michael E, Bomble Yannick J
1Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401 USA.
2Department of Genetics, University of Georgia, Athens, GA 30602 USA.
Biotechnol Biofuels. 2018 Feb 1;11:22. doi: 10.1186/s13068-018-1014-2. eCollection 2018.
Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, , was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, Cel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the exoproteome and how inter-molecular synergy between them confers with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms.
To advance the understanding of the exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even improve upon the activity of the whole exoproteome, even though some of the enzymes lack significant activity on their own.
We have demonstrated that it is possible to replicate the cellulolytic activity of the native exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.
嗜热微生物及其酶在工业应用中比嗜温微生物具有若干优势。例如，一种超嗜热厌氧菌最近从西伯利亚堪察加半岛的温泉中分离出来，显示出非常高的纤维素分解活性。此外，它是少数被认为是联合生物加工应用可行候选者的微生物之一。而且，该菌能够在无需酶或化学预处理的情况下解构植物生物质。这种能力是通过产生和分泌游离的、多模块和多功能的酶来实现的，其中一种酶，即Cel9A/Cel48A（也称为CelA），其性能优于商业酶制剂中的酶。此外，与商业真菌纤维素酶不同，该菌的完整外蛋白质组具有极高的热稳定性，并且在高温下具有高活性。因此，了解该菌外蛋白质组中酶的功能多样性以及它们之间的分子间协同作用如何赋予其高纤维素分解活性，是一项重要的工作，有助于生产更高效的生物质降解酶制剂，进而培育出更好的纤维素分解工业微生物。
为了增进对该菌外蛋白质组的了解，我们表达、纯化并测试了外蛋白质组中发现的四种主要酶，我们发现三种或四种表达量最高的酶组合表现出协同活性。我们还证明，这些酶的特定组合能够模拟甚至提高整个外蛋白质组的活性，尽管其中一些酶本身缺乏显著活性。
我们已经证明，利用最小基因集可以复制天然外蛋白质组的纤维素分解活性，并且这些最小基因集比整个外蛋白质组更具活性。未来，这可能会导致更简化、高效的纤维素分解酶制剂，或者当这些酶在为联合生物加工而设计的微生物中表达时提高产量。
