Optogenetics combines light and genetically-engineered photoreceptors to enable precise control of biological processes such as gene expression. There has been a recent surge of interest in utilizing optogenetics to manipulate microbial enzyme activity for metabolic engineering applications. Microbes transition from exponential growth to a non-growth stationary over the course of many metabolic fermentations. However, nearly all previous optogenetics studies have characterized microbial photoreceptors in exponential phase. Thus, it is imperative to engineer genetically-encoded photoreceptors that function robustly in stationary phase. One of the most highly utilized bacterial optogenetic systems is CcaSR, a green/red light photoreversible two-component system that utilizes the heme derivative phycocyanobilin (PCB) as a cofactor. Previously, we have engineered CcaSR strains with high dynamic ranges and fast dynamics in exponential growth. Here, we demonstrate that CcaSR loses it’s function in stationary phase. We then combine kinetic modeling, expression optimization of CcaSR, and PCB stabilization strategies to restore CcaSR dynamic range from 2-fold to over 400-fold in stationary phase. To achieve optical de-activation of gene expression in stationary phase, we further incorporate the mflon protease system into CcaSR. Finally, we utilize our stationary-phase optimized CcaSR system to optimize expression levels and dynamics of sts-TAL, an enzyme that converts L-tyrosine into the industrially relevant biosynthetic precursor, <i>p</i>-coumaric acid. Our work should facilitate the use of optogenetics for metabolic engineering and can be replicated for a wide range of additional two-component systems for other synthetic biology applications.

Engineering light-activated bacterial two-component systems to function in stationary phase

ACS Spring 2022

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