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

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.