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.
The project goal is to apply Protabit’s state-of-the-art computational protein design (CPD) platform, Triad, to improve the catalytic activity of a methane monooxygenase variant and show that it enables the commercial application of a biosynthetic gas-to-liquids pathway. After successfully improving the soluble expression of spmoB during Phase I, our Northwestern-Caltech-Protabit team is now well positioned to optimize activity and characterize the active species in molecular detail. Guided by the Phase I results, enzymatic activity optimization will proceed through iterative rounds of CPD and robotics-enabled screening so that only the most promising variants receive in-depth characterization. Interactions with a commercialization partner will guide later engineering cycles towards achieving process-oriented metrics, culminating in a laboratory scale demonstration of a biological gas-to-liquids process in our partner’s host organism. This Small Business Technology Transfer Phase II project aims to dramatically improve the catalytic activity of a recombinantly expressed methane monoxygenase (MMO) in order to fulfill the process demands of a biological gas-to-liquids pathway. The abundance of low-cost methane has stimulated strong interest in biological routes to convert methane into fuels and high-value chemicals. One strategy to address this opportunity is to transform MMO enzymes, which are native to methanotrophic bacteria and notoriously difficult to genetically manipulate, into industrially-relevant heterologous hosts. In Phase I, our Northwestern-Caltech-Protabit team engineered the particulate MMO (pMMO) catalytic subunit (spmoB) to allow it to be solubly expressed in E. coli, marking 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. This MMO construct will allow us to explore the biophysical properties and structure of spmoB, as well as to interrogate the mechanism of pMMO methane oxidation and address many unanswered questions in the MMO field. This information will directly facilitate our primary objective of engineering spmoB to increase its MMO activity under process-relevant conditions. High-performance MMO enzymes resulting from this work will then be employed as the primary step in a laboratory scale demonstration of methane conversion to isopropanol. If this research is successful, we will have applied methane monooxygenase (MMO) enzymes to enable a biological gas-to-liquids (GTL) pathway in an industrially-relevant host organism. Our high performing MMO would open up numerous new biological routes to chemicals and fuels from natural gas, capitalizing on abundant domestic shale gas reserves and reducing U.S. dependence on foreign oil. Furthermore, our enzyme technology has the potential to significantly decrease the capital cost of small-scale GTL, 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 our carbon footprint and spur domestic manufacturing, investment, and job creation. Furthermore, computational protein design (CPD)-based protein engineering methods can significantly reduce the costs of biotechnology R&D 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 discovery of functional end products. Furthermore, CPD-based protein engineering methods enable new products and technologies in myriad areas, including industrial enzymes and therapeutics. This project has the potential to promote U.S. economic prosperity and accelerate progress toward energy independence and environmental sustainability.
