467994 Characterization of Formaldehyde-Inducible Promoter with Massively Parallel Partitioning and Sequencing

Monday, November 14, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Julia R. Rohlhill, Nicholas R. Sandoval and Eleftherios T. Papoutsakis, Chemical and Biomolecular Engineering, University of Delaware, Newark, DE

Transcription factor-promoter pairs can be used to control expression of a desired gene based on a chemical input. Characterization of sequence-function relationships of DNA-protein interactions in transcription factors is key for understanding inducible regulator-promoter pairs. Nucleotide resolution quantitative analysis of these interactions enables the design of tunable synthetic promoters with varied and desirable properties.

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.


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See more of this Session: Poster Session: Bioengineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division