467994 Characterization of Formaldehyde-Inducible Promoter with Massively Parallel Partitioning and Sequencing
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 characterize the in vivo properties of the Pfrm/FrmR including the Hill cooperativity coefficient, sensitivity, and dynamic range for the wild type and ΔfrmR strain against varying formaldehyde concentrations using a fluorescent reporter plasmid and flow cytometry analysis. The constructed reporter system can induce more than 30-fold change in expression rate and is sensitive to formaldehyde concentrations ranging from 1 to 1000 µM, where transcriptional noise limits the lower end and toxicity the upper.
To elucidate the sequence-strength and sequence-repression relationships of the promoter regulator pair, we screened and partitioned a library of mutated promoters using FACS and analyzed the resulting activity-based binned populations with high-throughput sequencing. We quantitatively describe the effect of mutations at single nucleotide resolution 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 and the NIH through the NRSA F32GM109617.