STTR Phase I: Engineering a recombinant methane monooxygenase to convert methane to methanol for the production of fuels and chemicals

这个小型企业技术转让第一阶段项目旨在设计酶的变种，以促进甲烷完全生物转化为燃料和高价值化学品，如异丁醇和1，4-丁二醇。天然气的丰富和低成本激发了人们对开发生物合成途径实现这些转化的兴趣，来自甲烷营养细菌的甲烷单加氧酶(MMOS)可以通过将甲烷转化为甲醇来提供这一途径的第一步。然而，对甲烷氧化菌的基因操作是困难的？为了进行途径工程，需要一种可溶的、活性的重组表达的MMO。目前，唯一的重组MMO(SpmoB)以低产率和低活性表达。该项目旨在使用计算蛋白质设计(CPD)和高通量筛选来设计能够在重组宿主中可溶性表达的spmoB的活性变体。在第二阶段，这些改进的变体将作为进一步工程的平台，以增强MMO的催化活性。可溶表达的重组spmoB变体将极大地促进突变研究和结构表征，从而更好地了解对MMO活性、还原剂结合和底物特异性的要求。这项工作还应该阐明该家族中其他酶的机制，包括氨单加氧酶和相关的碳氢单加氧酶。这个项目的广泛影响/商业潜力是巨大的。如果成功，我们将开发一个用于工业用途的MMO优化平台。优化的MMO将减少肮脏、昂贵的化学催化剂的使用，并降低将搁置或燃烧的甲烷转化为甲醇的成本。提高甲烷氧化的成本效益将反过来降低异丁醇和1，4-丁二醇等下游产品的成本，并减少温室气体排放。全球天然气燃烧每年浪费约1000亿美元，向大气排放约3.6亿吨二氧化碳。通过促进搁浅或燃烧的甲烷转化为燃料和化学品，这项研究可以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，这项工作可能会进一步使用基于计算的蛋白质工程？这种方法可以通过将实验筛选工作转移到软件平台来降低研究成本。CPD输出的序列富含功能变体，可以加速发现新的/改进的蛋白质，并加快我们对蛋白质功能机制的理解。因此，该项目可以加深我们对MMOS和其他蛋白质的了解，并促进经济、能源和环境的可持续性。

This Small Business Technology Transfer Phase I project aims to engineer enzyme variants that will facilitate the complete bioconversion of methane into fuels and high-value chemicals such as isobutanol and 1,4-butanediol. The abundance and low cost of natural gas has stimulated interest in developing biosynthetic pathways to achieve these conversions, and methane monooxygenases (MMOs) from methanotrophic bacteria could provide the first step in such a pathway by converting methane to methanol. However, genetic manipulation of methanotrophic bacteria is difficult?a soluble, active recombinantly-expressed MMO is needed for pathway engineering. Currently, the only recombinant MMO (spmoB) expresses insolubly in low yields and with low activity. This project aims to use computational protein design (CPD) and high-throughput screening to engineer active variants of spmoB that are amenable to soluble expression in a recombinant host. In Phase II, these improved variants will serve as a platform for further engineering to enhance MMO catalytic activity. Solubly expressed recombinant spmoB variants will greatly facilitate mutagenesis studies and structural characterization, leading to a better understanding of requirements for MMO activity, reductant binding, and substrate specificity. This work should also shed light on mechanisms of other enzymes in this family including ammonia monooxygenases and related hydrocarbon monooxygenases. The broader impact/commercial potential of this project is substantial. If successful, we will have developed a platform for optimizing an MMO for industrial use. An optimized MMO would reduce the use of dirty, expensive chemical catalysts and decrease the cost of transforming stranded or flared methane into methanol. Improving the cost effectiveness of methane oxidation will in turn decrease the cost of downstream products such as isobutanol and 1,4-butanediol and diminish greenhouse gas emissions. Global gas flaring wastes ~$100 billion and emits ~360 million tons of CO2 into the atmosphere yearly. By facilitating the conversion of stranded or flared methane to fuels and chemicals, this research can decrease U.S. dependence on foreign oil, reduce our carbon footprint, and spur domestic manufacturing, investment, and job creation. In addition, this work may further the use of computational-based protein engineering?this approach can reduce research costs by shifting experimental screening efforts to the software platform. The sequences output by CPD are enriched in functional variants, which can accelerate the discovery of new/improved proteins and speed our understanding of the mechanisms involved in protein function. This project could thus further our understanding of MMOs and other proteins, and facilitate economic, energy, and environmental sustainability.