Universal platform for the bioconversion of single carbon feedstocks to fuels and chemicals

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There is increasing interest in developing more sustainable feedstocks for the chemicals and fuels. One-carbon compounds, including carbon dioxide and methane, are attractive alternative feedstocks for liquid fuel and chemicals production, because utilization of these abundant feedstocks can upgrade these low-value materials and at the same time lower greenhouse gas emissions. Photosynthetic microorganisms such as algae can use carbon dioxide to make fuels, and other microorganisms can metabolize methane, but not very efficiently. A more fundamental and efficient framework for metabolism of one-carbon compounds, designed at the molecular level from the bottom up, is needed to enable a viable biological process. The goal of this project is to use the tools of synthetic biology to engineer microorganisms to convert single carbon compounds, including carbon dioxide and methane, directly into a variety of chemicals and fuels. The educational activities associated with the project include the hosting and mentoring of Hispanic middle and high school students from the Houston Harmony Science in the laboratory of the principal investigator.The research will develop a new metabolic platform for the assimilation of single carbon compounds, including carbon dioxide or methane, directly into the production of organic chemicals and fuels within genetically engineered microorganisms. The proposed engineered platform defines a new metabolic architecture that consolidates carbon fixation, central metabolism, and product synthesis into a single pathway. The pathway uses formyl-CoA as a single carbon extension unit, which bypasses the need for the production of common metabolic intermediates and allows for elongation of a carbon backbone iteratively in single-carbon increments. The proposed platform is inspired by the catabolic alpha-oxidation pathway, and uses the enzyme hydroxyl-acyl-CoA lyase to catalyze C-C bond formation between formyl-CoA and an aldehyde. The engineered platform consists of three modules, including an activation module that converts a single carbon molecule to an extender unit, an elongation module in which the extender unit is added to a carbon backbone, and a termination module, which converts intermediates of the elongation module to target products. There are four research objectives to develop this pathway. The first objective is to rationally design a pathway allowing for the iterative elongation of a carbon backbone using 1-carbon extender unit formyl-CoA and the synthesis of longer-chain products from the intermediates of this pathway. The second objective involves the in vitro characterization of pathway components and functional assembly of individual modules using molecular and biochemical techniques. The third objective focuses on the in vivo construction and characterization of the pathway to achieve product synthesis via microbial fermentations, and the fourth objective focuses on a system-wide characterization of wild-type and engineered strains. Toward this end, functional components for each enzymatic step will be assembled in vivo by making use of synthetic biology tools to facilitate the efficient conversion of single carbon feedstocks to valuable products via microbial fermentation. The new C-C bond formation mechanism advanced by this research will be expected to make fundamental and potentially impactful contributions to one-carbon metabolism and biosynthesis.