STTR Phase I: Engineering Polysaccharide Monooxygenases for Enhanced Sugar Recovery From Biomass

这个小型企业技术转移（STTR）一期项目旨在应用计算蛋白设计（CPD）来设计多糖单加氧酶（PMOs），以提高热稳定性，这样PMOs和关键纤维素酶的混合物将更有效地将木质纤维素生物质转化为可发酵糖。第一阶段的目标是：(a)评估几种天然PMO的表达、热稳定性、PMO活性和增强纤维素酶混合物的纤维素分解活性的能力，然后选择一种或多种作为工程目标；(b)使用CPD设计pmo以提高热稳定性；(c)实验筛选设计的PMO变体库；(d)通过额外的工程设计来提高最佳命中弹的性能。本文提出的工作将有助于阐明PMOs在其他纤维素酶的情况下在纤维素分解降解中的作用。这个项目可能会进一步加深我们对这些酶的底物特异性的认识。任何可能被解决的新的晶体结构也可以增强我们对该酶家族的结构差异和底物特异性的理解，并可能有助于阐明结构-功能关系。预期的技术结果是一种或多种PMO变体，相对于野生型PMO，其热稳定性提高了5-10°C。如果成功，该项目的更广泛的影响/商业潜力将是：(a)降低将生物质转化为单糖的成本，单糖是生产可再生燃料和化学品的主要原料；(b)鼓励更广泛地使用酶水解，这是一种可持续和环保的过程；(c)展示我们蛋白质工程平台的实用性。通过促进以生物为基础的乙醇生产、先进的生物燃料和其他石油衍生材料的替代品，这项研究可以帮助减少美国对外国石油的依赖，减少我们的碳足迹，并刺激国内制造业、投资和创造就业机会。此外，从纤维素中获取糖可以通过鼓励种植能够在不适合粮食生产的边缘土地上生长的专用原料作物来遏制粮食与燃料的争论。基于cpd的蛋白质工程方法通过将大量的实验筛选工作转移到软件平台上，可以显着降低生物学研究的成本。CPD设计的库输出通常具有丰富的功能变体，从而加快了功能性最终产品的交付。此外，基于cpd的蛋白质稳定方法在包括工业酶和治疗学在内的众多领域实现了新产品和新技术。这个项目将加速美国在经济、能源和环境可持续性方面的进展。

This Small Business Technology Transfer (STTR) Phase I project aims to apply computational protein design (CPD) to engineer polysaccharide monooxygenases (PMOs) for improved thermostability, so that a cocktail of PMOs and key cellulases will more efficiently convert lignocellulolosic biomass to fermentable sugars. The objectives of Phase I are to: (a) evaluate several natural PMOs for expression, thermostability, PMO activity, and ability to enhance the cellulolytic activity of a cellulase cocktail, and then select one or more as targets for engineering; (b) use CPD to design the PMOs for improved thermostability; (c) experimentally screen libraries of designed PMO variants; and (d) improve the properties of best hits with additional rounds of engineering. The work proposed here will help elucidate the role PMOs play in cellulolytic degradation in the context of other cellulases. This project may further our knowledge of the substrate specificities of these enzymes. Any novel crystal structures that may be solved could also enhance our understanding of the structural differences and substrate specificities within this family of enzymes and may serve to elucidate structure-function relationships. The anticipated technical result is one or more PMO variants that have increased thermostability of 5-10°C relative to the wild-type PMO. The broader impact/commercial potential of this project, if successful, will be to: (a) reduce the costs of converting biomass into simple sugars, which are a principal raw material for the production of renewable fuels and chemicals, (b) encourage the broader use of enzymatic hydrolysis, a sustainable and environmentally-friendly process, and (c) demonstrate the utility of our protein engineering platform. By facilitating the bio-based production of ethanol, advanced drop-in biofuels, and substitutes for other petroleum-derived materials, this research can help reduce U.S. dependence on foreign oil, reduce our carbon footprint, and spur domestic manufacturing, investment, and job creation. In addition, sourcing sugars from cellulose can curb the food-versus-fuel debate by encouraging the farming of dedicated feedstock crops capable of growing on marginal lands unsuitable for food production. CPD-based protein engineering methods can significantly reduce the costs of biological research by shifting a significant amount of experimental screening effort to the software platform. The designed libraries output by CPD are typically enriched in functional variants, accelerating the delivery of functional end-products. Furthermore, CPD-based protein stabilization methods enable new products and technologies in myriad areas, including industrial enzymes and therapeutics. This project will accelerate U.S. progress toward economic, energy, and environmental sustainability.