STTR Phase II: Development of a computational protein engineering platform and its application to methane activating enzymes

这个小企业创新研究第二阶段项目的更广泛的影响/商业潜力将联合收割机计算蛋白质工程（CPE）软件，工具和方法结合成一个“平台技术”，使新产品和技术在广泛的科学领域，包括工业酶，制药，治疗，医疗诊断和生物能源。该项目将在具有商业和环境价值的应用中验证CPE平台：利用甲烷作为原料的工程酶。 开发的工程甲烷活化酶将开辟许多新的生物途径，从天然气中获得化学品和燃料，利用国内丰富的页岩气储量，提高美国的能源独立性。 这种CPE酶技术有可能显着降低小规模天然气液化（GTL）工厂的资本成本，从而实现天然气的捕获和货币化。 在北达科他州，超过四分之一的天然气被燃烧或排放，浪费了宝贵的自然资源，并不必要地排放温室气体。 通过促进将滞留或燃烧的甲烷转化为燃料和高价值化学品，这项研究可以帮助减少碳足迹并刺激国内制造业、投资和创造就业机会。第二阶段项目的目标是开发和许可CPE平台技术，使实验生物科学家能够利用计算蛋白质设计和工程的力量;通过工程设计具有增加的可溶性表达和甲烷氧化活性的新型甲烷活化酶来验证CPE平台技术;并与该技术的潜在许可证持有者合作，努力将工程酶纳入其工业宿主生物体中。这些生物将能够使用甲烷作为原料，用于生物生产各种终端产品，包括液体运输燃料，商品化学品和高价值精细化学品。在第一阶段，西北-加州理工学院-Protabit团队成功地改造了颗粒MMO（pMMO）催化亚基（spmoB），使其能够在E.杆菌 这是第一次证明活性甲烷氧化酶可以在遗传上易处理的重组宿主中以显著量可溶性表达和纯化。 第二阶段将继续这项工作，目标是通过提高spmoB酶的活性和解决将其插入工业宿主的某些其他挑战来使其更有效。

The broader impact/commercial potential of this Small Business Innovation Research Phase II project will combine computational protein engineering (CPE) software, tools and methods into a "platform technology" that enables new products and technologies in a wide range of scientific areas, including industrial enzymes, pharmaceuticals, therapeutics, medical diagnostics and bioenergy. This project will validate the CPE platform in an application that has both commercial and environmental value: engineered enzymes for utilizing methane as a feedstock. The engineered methane activating enzymes developed will open up numerous new biological routes to chemicals and fuels from natural gas, capitalizing on abundant domestic shale gas reserves and improving U.S. energy independence. This CPE enzyme technology has the potential to significantly decrease the capital cost of small-scale Gas-to-liquid (GTL) plant, enabling the capture and monetization of otherwise economically-stranded natural gas. In North Dakota, more than a quarter of the total gas produced is flared or vented, wasting a valuable natural resource and needlessly emitting greenhouse gases. By facilitating the conversion of stranded or flared methane to fuels and high-value chemicals, this research can help reduce carbon footprint and spur domestic manufacturing, investment, and job creation. The objectives of this Phase II project are to develop and license a CPE Platform technology that will enable experimental biological scientists to harness the power of computational protein design and engineering; to validate the CPE Platform technology by engineering novel methane activating enzymes that have increased soluble expression and methane-oxidizing activity; and in collaboration with potential licensees of this technology, work to incorporate the engineered enzyme into their industrial host organisms. These organisms will be enabled to use methane as a feedstock for the bio-production of a wide range of end products, including liquid transportation fuels, commodity chemicals and high value fine chemicals. In Phase I, the Northwestern-Caltech-Protabit team succeeded in engineering the particulate MMO (pMMO) catalytic subunit (spmoB) to allow it to be solubly expressed in E. coli. This was the first demonstration of an active methane-oxidizing enzyme that can be solubly expressed and purified in significant quantities in a genetically-tractable recombinant host. Phase II will continue this work, with the objective of making the spmoB enzyme more effective by increasing its activity and solving certain other challenges for inserting it into an industrial host.