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

植物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。它由葡萄糖分子链组成。 纤维素酶是将这些链切割成其糖构建块的酶。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生物燃料。该项目的目的是使用尖端的光学显微镜来更好地了解纤维素酶的工作原理。 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，将编写和提供教育材料，让植物生物学学生接触单分子生物物理学，让生物物理学学生接触植物生物学。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覆纤维素，从而阻止纤维素酶的进入。本计画将结合联合收割机、单分子萤光追踪、计算模拟及蛋白质工程等技术，探讨纤维素酶的作用机制。拟议的实验建立在正在进行的工作中，研究人员构建了一个定制的多模态显微镜，并用它来跟踪纤维素酶沿沿着纤维素移动纳米级精度。这项工作分为三个目的，探索纤维素酶活性的具体方面。目的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。目标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过早的底物释放。目的3的目标是确定纤维素酶导航生物聚合物的复杂混合物的机制，同时实现其纤维素酶底物的特异性消化，避免脱靶结合，该奖项反映了NSF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进行评估，被认为值得支持的搜索.

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.