(509b) Metabolite-Responsive Transcription Factors for Feed Activated Pathway Expression in E. coli

Metabolic engineering of microbes enables the production of valuable chemicals by introducing heterologous enzymes to the recombinant cell. However, it is well known that the introduced enzymes impose a metabolic burden to the host. In response, common fermentation practice allows cells to dedicate resources to biomass generation before expressing heterologous enzymes from chemically inducible promoters, such as the lac promoter and its IPTG inducer. While chemically inducible promoters have been widely adopted due to their simplicity and pathway-independent nature, they introduce a chemical extrinsic to the process, and the associated cost. We developed a pathway specific expression control strategy that utilizes the galacturonate feed as the inducer, thereby eliminating the need for extrinsic chemicals. Our strategy utilizes a galacturonate-responsive transcription factor to express heterologous enzymes for a glycerate production pathway developed in our lab. We constructed variants of a galacturonate biosensor with a heterologous transcription factor and cognate hybrid promoter, and selected for the best performer through fluorescence characterization. We showed that native E. coli regulatory systems have limited interaction with our biosensor and demonstrated simple strain engineering that enhanced biosensor performance. The selected galacturonate biosensor variant was used to express our glycerate production pathway in a feed dependent manner. We confirmed that heterologous pathway enzyme expression is induced in the presence of the galacturonate feed through qRT-PCR. Our control circuit replaced the IPTG induction that was previously used on the glycerate production pathway with no sacrifice to glycerate titer or growth rate. Our work demonstrates that feed activated pathway expression can be used to remove extrinsic chemical inducers from a microbial production system.