Curated Optogenetic Publication Database

Abstract: Dynamic metabolic engineering is a strategy to switch key metabolic pathways in microbial cell factories from biomass generation to accumulation of target products. Here, we demonstrate that optogenetic intervention in the cell cycle of budding yeast can be used to increase production of valuable chemicals, such as the terpenoid β-carotene or the nucleoside analog cordycepin. We achieved optogenetic cell-cycle arrest in the G2/M phase by controlling activity of the ubiquitin-proteasome system hub Cdc48. To analyze the metabolic capacities in the cell cycle arrested yeast strain, we studied their proteomes by timsTOF mass spectrometry. This revealed widespread, but highly distinct abundance changes of metabolic key enzymes. Integration of the proteomics data in protein-constrained metabolic models demonstrated modulation of fluxes directly associated with terpenoid production as well as metabolic subsystems involved in protein biosynthesis, cell wall synthesis, and cofactor biosynthesis. These results demonstrate that optogenetically triggered cell cycle intervention is an option to increase the yields of compounds synthesized in a cellular factory by reallocation of metabolic resources.

Abstract: In biotechnological protein production processes, the onset of protein unfolding at high gene expression levels leads to diminishing production yields and reduced efficiency. Here we show that in silico closed-loop optogenetic feedback control of the unfolded protein response (UPR) in S. cerevisiae clamps gene expression rates at intermediate near-optimal values, leading to significantly improved product titers. Specifically, in a fully-automated custom-built 1L-photobioreactor, we used a cybergenetic control system to steer the level of UPR in yeast to a desired set-point by optogenetically modulating the expression of α-amylase, a hard-to-fold protein, based on real-time feedback measurements of the UPR, resulting in 60% higher product titers. This proof-of-concept study paves the way for advanced optimal biotechnology production strategies that diverge from and complement current strategies employing constitutive overexpression or genetically hardwired circuits.

Abstract: Dynamically controlling cellular metabolism can improve a cell's yield and productivity towards a target compound. However, the application of this strategy is currently limited by the availability of reversible metabolic switches. Unlike chemical inducers, light can readily be applied and removed from the medium multiple times without causing chemical changes. This makes light-inducible systems a potent tool to dynamically control cellular metabolism. Here we describe the construction of a light-inducible metabolic switch to regulate flux distribution between two glycolytic pathways, the Embden-Meyerhof-Parnas (EMP) and oxidative pentose phosphate (oxPP) pathways. This was achieved by using chromatic acclimation sensor/regulator (CcaSR) optogenetic system to control the expression of pgi, a metabolic gene which expression determines flux distribution between EMP and oxPP pathways. Control over these pathways may allow us to maximize Escherichia coli's yield on highly-reduced compounds such as mevalonate. Background pgi expression of the initial CcaSR construct was too high to significantly reduce pgi expression during the OFF-state. Therefore, we attenuated the system's output leakage by adjusting plasmid copy number and by tagging Pgi with ssRA protein degradation signal. Using our CcaSR-pgi ver.3, we could control EMP:oxPP flux ratio to 50:49 and 0.5:99 (of total glycolytic flux) by exposure to green and red light, respectively.