About This Project

The soluble methane monooxygenase (sMMO) enzyme catalyzes the oxygen dependent conversion of methane to methanol. The recombinant expression of sMMO in Escherichia coli and Komagataella phaffii will enable these industrial biotechnology relevant model organisms to capture a greenhouse gas and make methanol, a liquid fuel. The natural methylotrophy found in K.phaffii and the addition of synthetic methylotrophy in E.coli can make value added products from the trapped carbon.

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Motivating Factor

Methane is a potent greenhouse gas that is exacerbating global warming through the increasing atmospheric levels from agriculture, animal livestock, and oil/natural gas sources [1,2]. At the same time, it is a valuable energy source that is wasted from small-scale sources such as oil-fields and landfills through flaring and venting [3]. This motivates the design of a chemical and/or biochemical process to reduce global warming through capturing methane whilst making value-added compounds from the trapped carbon such as methanol, which is a liquid fuel and chemical industry feedstock [4]. Alternatively, the captured methane can be utilized as a carbon source for industrial microbial biotechnology in lieu of sugars that compete with the human food supply [5].

Specific Bottleneck

The key challenge lies in the functionalization of the strong C-H bond of methane, which has been overcome by soluble and particulate methane monooxygenase enzymes (sMMO, pMMO) that convert methane to methanol under ambient conditions [6]. Methanotrophs are non-model organisms with a constrained capability for genetic manipulation [7]. Heterologously expressing these enzymes in model organisms possessing either synthetic or natural methylotrophy (E. coli and K. phaffii) could enable a biomanufacturing process to capture methane and make value-added products [8,9,10,11]. However, the specific bottleneck towards this goal is the inability to recombinantly express sMMO/pMMO in an active form. For sMMO, this stems from the insolubility of the active site containing hydroxylase protein (MMOH) [12,13,14]. This has stymied structure-function studies of MMOH and protein engineering of sMMO, which is a key requirement to overcome specific constraints in two other problem statements [15,16].

Actionable Goals

There has been recent progress in improving MMOH solubility in E.coli, which has enabled sMMO-mediated cellular methane oxidation in a preprint report [12,13,14]. This advance should be built upon to engineer robust sMMO activity in E.coli and K.phaffii. Methanotrophs display methane uptake rates (V_max) of 10 – 31 mmoles.g_CDW^-1.h^-1 while expressing sMMO and pMMO enzymes at levels upto 20 % of total protein content [17,18,19,20]. Therefore, a reasonable first target for heterologous sMMO expression can be ~ 4 – 8 mmoles.g_CDW^-1.h^-1 for methane capture. This would provide the necessary impetus for the scientific community to combine this expression construct with the genetic modules enabling synthetic methanotrophy in E.coli [8,9,10].