Characterization and Engineering of a Formaldehyde-Responsive Promoter-Regulator System for Synthetic Methylotrophy and Beyond

Understanding the quantitative sequence-function relationship in DNA is essential for furthering synthetic biology design principles. Transcription factors use protein-DNA interactions to regulate gene expression. Elucidation of these specific interactions is key for further engineering a range of inducible and tunable promoters.

Applications where engineered pathways produce toxic intermediates (e.g. formaldehyde) require dynamic internal pathway balancing to avoid growth and production inhibition. The formaldehyde-inducible promoter (Pfrm) transcribing the frmRAB operon is repressed in the presence of FrmR, the product of the first gene in the operon. FrmA and FrmB encode the glutathione-dependent formaldehyde dehydrogenase and S-formylglutathione hydrolase, respectively, which together oxidize formaldehyde to carbon dioxide. While the general locus of the Pfrm-FrmR interaction has been identified, the consensus sequence has not been previously elucidated.

Here, we describe the sensitivity, dynamic range, and noise of the response of the Pfrm promoter to varying levels of formaldehyde with construction of a GFP reporter plasmid and flow cytometry analysis. The system is sensitive to formaldehyde concentrations ranging from 10 to 1000 µM, where toxicity limits the upper end. We employed this formaldehyde reporter plasmid to successfully isolate functional heterologous methanol dehydrogenase genes in methanol-supplemented media using fluorescence-activated cell sorting (FACS).

To elucidate the sequence-strength and sequence-repression relationships of the promoter regulator pair, we screened a mutated library of synthetically-derived “mPfrm” promoters using FACS and analyzed the resulting activity-based binned populations with high throughput sequencing. Using information theory principles, we quantitatively describe the effect of mutations on promoter strength and efficacy of the repressor. This process enables a generalizable method for construction of custom tunable inducible promoter systems for refactoring pathways for precise metabolic flux control.

This work was supported by the US DOE ARPA-E agency through contract no. DE-AR0000432.