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

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Precise control of gene expression is essential for synthetic biology applications.
• Understanding promoter sequence-function relationships is key for inducible gene expression systems.
• Sort-seq provides a rapid and quantitative method for analyzing these relationships.

Purpose of the Study:

• To elucidate the sequence-function relationship of the formaldehyde-inducible Escherichia coli promoter (P_frm) at single-nucleotide resolution.
• To engineer improved formaldehyde-inducible promoters with enhanced dynamic range and tunable expression.
• To demonstrate the utility of engineered promoters in driving heterologous gene expression for metabolic engineering.

Main Methods:

• A massively parallel sort-seq approach was employed.
• A library of mutated P_frm promoters was created and cloned upstream of gfp.
• Fluorescence-activated cell sorting (FACS) and high-throughput sequencing were used to analyze promoter variants based on GFP expression levels.

Main Results:

• Two repressor binding sites within the P_frm were identified and their sequence-function relationships characterized.
• Key mutations were identified to tune promoter activity, leading to engineered variants with significantly reduced basal expression and increased induced expression.
• Engineered promoters exhibited up to a 3.6-fold stronger response (relative dynamic range).

Conclusions:

• The study successfully mapped the sequence-function landscape of the formaldehyde-inducible promoter.
• Engineered promoters provide predictable and tunable gene expression control.
• The application of an engineered promoter to drive heterologous methanol assimilation genes resulted in increased biomass production on methanol, highlighting its potential in metabolic engineering.