Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol

• Published in: ACS synthetic biology 2017-08, Vol.6 (8), p.1584-1595
• Main Authors: Rohlhill, Julia, Sandoval, Nicholas R, Papoutsakis, Eleftherios T
• Format: Article
• Language: English
• Subjects: Algorithms; BASIC BIOLOGICAL SCIENCES; biosensor; Cell Proliferation - genetics; Escherichia coli - cytology; Escherichia coli - drug effects; Escherichia coli - physiology; Escherichia coli Proteins - genetics; Flow Cytometry - methods; Formaldehyde - administration & dosage; Gene Expression Regulation, Bacterial - drug effects; Gene Expression Regulation, Bacterial - genetics; High-Throughput Nucleotide Sequencing - methods; Metabolic Engineering - methods; Methanol - metabolism; promoter engineering; Promoter Regions, Genetic - genetics; sort-seq; transcription factor binding; Transcriptional Activation - genetics; transcriptional fine-tuning
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.7b00114

Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence–function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence–function relationships in a robust and rapid way. Here we utilized a massively parallel sort-seq approach to analyze the formaldehyde-inducible Escherichia coli promoter (P frm ) with single-nucleotide resolution. A library of mutated formaldehyde-inducible promoters was cloned upstream of gfp on a plasmid. The library was partitioned into bins via FACS on the basis of green fluorescent protein (GFP) expression level, and mutated promoters falling into each expression bin were identified with high-throughput sequencing. The resulting analysis identified two 19 base pair repressor binding sites, one upstream of the −35 RNA polymerase (RNAP) binding site and one overlapping with the −10 site, and assessed the relative importance of each position and base therein. Key mutations were identified for tuning expression levels and were used to engineer formaldehyde-inducible promoters with predictable activities. Engineered variants demonstrated up to 14-fold lower basal expression, 13-fold higher induced expression, and a 3.6-fold stronger response as indicated by relative dynamic range. Finally, an engineered formaldehyde-inducible promoter was employed to drive the expression of heterologous methanol assimilation genes and achieved increased biomass levels on methanol, a non-native substrate of E. coli.