Enhancing cellulase activity through single-molecule imaging and protein engineering as a testbed for understanding and improving enzymatic deconstruction of insoluble substrates

植物来源的纤维素是地球上最丰富的生物聚合物。纤维素用于造纸、制衣和建筑材料。它是由葡萄糖分子链组成的。纤维素酶是将这些链切割成糖基的酶。减少我们对化石燃料依赖的一种方法是将大量的非食用植物材料，如柳枝稷和废弃的玉米秸秆，消化成糖分子。这些可以发酵成可再生的生物燃料。这个项目的目标是使用尖端的光学显微镜来更好地了解纤维素酶是如何工作的。所产生的知识将用于设计和测试可能更有效的酶，降低成本并增加可再生能源的可用性。在研究的同时，将开发和提供教育材料，使植物生物学的学生了解单分子生物物理学，并使生物物理学的学生了解植物生物学。为了加强和增加生物经济劳动力的多样性，将积极招募来自代表性不足人群的学生参与研究项目。纤维素酶分解植物细胞壁的能力受到纤维素结晶度的阻碍。其他聚合物如木质素和木聚糖的存在被认为包裹了纤维素，从而阻止了纤维素酶的进入。该项目将结合单分子荧光跟踪、计算建模和蛋白质工程来研究纤维素酶的机制。这项实验是建立在研究人员正在进行的工作的基础上的，他们建造了一个定制的多模态显微镜，并用它来追踪纤维素酶沿着纤维素运动的纳米级精度。这项工作分为三个目的，探索纤维素酶活性的具体方面。Aim 1的目标是揭示调节底物结合和纤维素聚合物链进入纤维素酶的催化通道以启动纤维素解构的关键原理。Aim 2的目标是揭示如何调整纤维素酶的底物亲和力和周转率，以最大限度地提高酶的催化活性，同时最大限度地减少产物抑制和底物过早释放。Aim 3的目标是确定纤维素酶在复杂的生物聚合物混合物中导航的机制，同时实现纤维素酶底物的特异性消化，避免脱靶结合，克服细胞壁复杂性造成的阻碍纤维素降解的障碍。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Plant-derived cellulose is the most abundant biopolymer on Earth. Cellulose is used to make paper, clothing, and building materials. It is composed of chains of glucose molecules. Cellulases are enzymes that cut these chains into their sugar building blocks. One approach for reducing our reliance on fossil fuels is to digest abundant, non-food plant materials like switchgrass and discarded corn stalks into sugar molecules. These can be fermented into renewable biofuels. The objective of this project is to use cutting-edge light microscopy to better understand how cellulases work. The knowledge generated would be used to design and test enzymes that may be more efficient, lowering the cost and increasing the availability of renewable energy. In parallel with research, educational materials to expose plant biology students to single-molecule biophysics and biophysics students to plant biology will be developed and delivered. To strengthen and increase the diversity of the bioeconomy workforce, students from underrepresented populations will be actively recruited to participate in the research project.The ability of cellulases to break down plant cell walls is hindered by the crystallinity of cellulose. The presence of other polymers such as lignin and xylans are thought to coat cellulose and thus block access by cellulase enzymes. This project will combine single-molecule fluorescence tracking, computational modeling, and protein engineering to investigate cellulase mechanisms. The proposed experiments build on ongoing work in which the investigators constructed a custom multimodal microscope and used it to track cellulases moving along cellulose with nanometer-scale precision. The work is divided into three Aims that explore specific aspects of cellulase activity. The goal of Aim 1 is to uncover the key principles that regulate substrate binding and threading of the cellulose polymer strand into the catalytic tunnel of the cellulase enzyme to initiate cellulose deconstruction. The goal of Aim 2 is to uncover how the substrate affinity and turnover rate of cellulases are tuned to maximize the enzyme’s catalytic activity while minimizing product inhibition and premature substrate release. The goal of Aim 3 is to identify mechanisms by which cellulases navigate complex mixtures of biopolymers, while achieving specific digestion of their cellulase substrate, avoiding off-target binding, and overcoming roadblocks imposed by cell wall complexity that hamper cellulose degradation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.