In situ product extraction using the sustainable solvent supercritical carbon dioxide is a promising method to relieve end-product toxicity with potential decreased purification costs. Previously, we have engineered an environmental-isolate of Bacillus megaterium called SR7 that is tolerant of the harsh supercritical carbon dioxide conditions to produce short-chained, branched alcohols that are well extracted by the solvent. While initial attempts at engineering SR7 were successful, the limited genetic tools to further modify the organism hindered metabolic engineering progress, especially the lack of strong, constitutive promoters. To expand the synthetic toolbox for the microbe, promoter mining was undertaken from an RNAseq library for B. megaterium SR7 strain grown under a variety of growth conditions, including aerobically, anaerobically under low pressures of carbon dioxide, and under supercritical carbon dioxide. Using gene expression results, several promoters were selected that showed high-level transcription under all growth conditions for their general use in engineering this strain. Promoters were assessed by expressing green fluorescent protein (GFP) under different growth conditions and medium combinations. The promoter related to polyhydroxyalkanoate production, phaR, was found to have high, constitutive expression, comparable to the widely utilized B. megaterium xylose-inducible promoter in both strength and rapidity in minimal medium. Other promoters related to citrate synthesis and polyhydroxyalkanoate synthesis had moderate expression, permitting fine-tuned transcription. Work is underway to test these promoters under other growth conditions and within the context of biological production of alcohols. This research is broadly applicable to the growing B. megaterium engineering community, and the universality of these promoters is being explored in different strains.

Expanding the promoter set for a super-critical CO2 tolerant strain of B. megaterium

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.