Development of Genetic Tools for the Metabolic Engineering of the Thermophilic Acetogen Moorella Thermoacetica

Synthesis gas (syngas) fermentation by anaerobic bacteria is a novel method for the biological production of fuels and chemicals from gaseous substrates such as carbon monoxide (CO), carbon dioxide (CO₂), and hydrogen (H₂). These substrates are renewable, produced via biomass gasification or natural gas reformation, thus the biological gas-to-liquids (GTL) process represents a sustainable platform that will be important for reducing dependence on fossil fuels. However, the range of products available from syngas fermentation, as exemplified by the commercial processes of LanzaTech, Coskata and INEOS, is currently limited by the absence of robust genetic engineering tools for the organisms capable of metabolizing the gases in question. We present here our progress in developing these tools to enable the metabolic engineering of the acetogen Moorella thermoacetica. This thermophilic, gram-positive anaerobe naturally produces acetate from H₂/CO₂ or CO via the canonical Wood-Ljungdahl pathway in order to provide the energy required for cell growth. As a homoacetogen, producing only acetate as its metabolic byproduct, Moorella is an ideal candidate for a production platform, but stable transformation of this organism has only recently been reported, and more development is required to establish a set of tools for convenient, tunable heterologous expression. To that end, we describe the development of a Flow Cytometry assay to optimize DNA delivery conditions, as well as our progress improving the robustness of the transformation protocol and expressing heterologous genes.