# A stationary phase-specific bacterial green light sensor for enhancing metabolite production

## TL;DR

Researchers developed a bacterial green light sensor that works during stationary phase, improving the production of valuable metabolites.

## Contribution

A new stationary phase-specific bacterial green light sensor (CcaSRstat) was engineered for enhanced metabolite production.

## Key findings

- CcaSRstat imposes low metabolic burden and has an 80-fold green light response in stationary phase.
- Pulsatile light with CcaSRstat increased production of p-Coumaric acid and betaxanthin pigments.
- The system functions reliably in benchtop bioreactor conditions.

## Abstract

Genetically-encoded sensors are used to control protein and metabolite production in bacterial fermentations. However, these sensors are generally optimized for exponential growth rather than stationary phase where production occurs. Here, we find that our previously engineered E. coli green light sensor CcaSR, which functions robustly in exponential phase, fails in stationary phase due to spontaneous loss of an engineered chromophore biosynthetic pathway and accumulation of CcaS and CcaR. We optimize the genetic context and expression determinants of each component, resulting in a stable system named CcaSRstat that imposes little metabolic burden, exhibits low leakiness and an 80-fold green light response, and functions exclusively in stationary phase. We combine CcaSRstat-driven enzyme expression with varied static and periodic illumination patterns to achieve high titers of the industrially-relevant phenylpropanoid p-Coumaric acid and demonstrate that these optimizations scale to benchtop bioreactor conditions. Finally, we use CcaSRstat to optimize the expression level of a co-transcribed multi-enzyme metabolic pathway encoding production of plant-derived betaxanthin family pigments. Stationary phase-optimized bacterial sensors should enhance fermentation productivity by enabling rapid interrogation of the impact of enzyme expression level and induction dynamics.

Genetically encoded sensors are generally optimized to function during exponential growth rather than stationary phase, which limits their potential value for metabolic engineering and bioproduction. Here, authors engineer a stationary phase green light sensor and use pulsatile light to optimize production of industrially relevant small molecules.