Programmable gene regulation for metabolic engineering using decoy transcription factor binding sites

Abstract

Transcription factor decoy binding sites are short DNA sequences that can serve as "sponges" to titrate a transcription factor away from its natural binding site, therefore regulating gene expression. In this study, we harness decoy sites to develop synthetic transcription factor sponge systems to regulate gene expression for metabolic pathways in Escherichia coli. We show that transcription factor sponges can effectively regulate expression of native and heterologous genes. Tunability of the sponge can be engineered via changes in copy number or modifications to the DNA decoy site sequence. Using arginine biosynthesis as a showcase, we observe a 16-fold increase in arginine production when we introduce the sponge system to steer metabolic flux towards increased arginine biosynthesis, with negligible growth differences compared to the wild type strain. The sponge-based production strain shows high genetic stability; in contrast to a gene knock-out approach where mutations were common, we detected no mutations in the production system using the sponge-based strain. We further show that transcription factor sponges are amenable to multiplexed library screening by demonstrating enhanced tolerance to pinene with a combinatorial sponge library. Our study shows that transcription factor sponges are a powerful and compact tool for metabolic engineering.