Curated Optogenetic Publication Database

Abstract: Dynamic metabolic engineering enables temporal redirection of microbial metabolism from biomass production to product synthesis. Here, we show that optogenetic control of protein kinase A (PKA) activity via light-regulated modulation of intracellular cyclic AMP (cAMP) levels can enhance heterologous production of β-carotene and cordycepin in Saccharomyces cerevisiae. To enable exclusive, glucose-independent control of cAMP synthesis, the photoactivatable adenylyl cyclase bPAC from Beggiatoa sp. was introduced into cells lacking the endogenous adenylyl cyclase Cyr1 or with lowered Cyr1 levels using an optogenetically controlled degron. Despite being growth-competent under illumination, the bPAC-containing yeast strain showed alterations in energy metabolism under all conditions. Quantitative proteome analysis using timsTOF mass spectrometry revealed profound changes in central carbon metabolism, sulfur homeostasis, energy charge, and ribosome biogenesis upon uncoupling cAMP from nutrient-dependent regulation, particularly under sustained light activation. These results highlight the critical role of dynamic Cyr1-dependent regulation for central metabolism, and underscore the biotechnological promise of refined PKA-targeted strategies for eukaryotic cell factories.IMPORTANCECarbon-footprint-minimized production of fine chemicals, pharmaceuticals, and biofuels requires optimized microbial cell factories with tailored metabolic performance. We employed optogenetic dynamic metabolic engineering in baker's yeast by uncoupling nutrient sensing from cAMP signaling using a light-controlled adenylate cyclase. Precise light regulation of intracellular cAMP levels and PKA activity enabled acute control of the metabolism, redirecting resources toward product synthesis, and boosting the production of valuable compounds such as β-carotene and cordycepin. Quantitative proteomics revealed that uncoupling of the cAMP-PKA axis from glucose sensing profoundly reprograms the central carbon metabolism and other key cellular processes. This approach provides a blueprint for refined, light-tunable strategies targeting the cAMP-PKA axis directly with light, e.g., for enhanced bioethanol production. Moreover, our data provide evidence for the profound influence of the cAMP-PKA axis on metabolism and balanced energy production that are fundamental for efficient production in microbial cell factories.

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: Optogenetic tools have been proven to be useful in regulating cellular processes via an external signal. Light can be applied with high spatial and temporal precision as well as easily modulated in quantity and quality. Natural photoreceptors of the light oxygen voltage (LOV) domain family have been characterized in depth, especially the LOV2 domain of Avena sativa (As) phototropin 1 and its derivatives. Information on the behavior of LOV2 variants with changes in the photocycle or the light response has been recorded. Here, we applied well-described photocycle mutations on the AsLOV2 domain of a photosensitive transcription factor (psTF) as well as its variant that is part of the photosensitive degron (psd) psd3 in Saccharomyces cerevisiae. In vivo and in vitro measurements revealed that each photoreceptor component of the light-sensitive transcription factor and the psd3 module can be modulated in its light sensitivity by mutations that are known to prolong or shorten the dark-reversion time of AsLOV2. Yet, only two of the mutations showed differences in the in vivo behavior in the context of the psd3 module. For the AsLOV2 domain in the context of the psTF, we observed different characteristics for all four variants. Molecular dynamics simulations showed distinct influences of the shortened Jα helix and the V416L mutation in the context of the psd3 photoreceptor. In conclusion, we demonstrated the tunability of two optogenetic tools with a set of mutations that affect the photocycle of the inherent photoreceptors. As these optogenetic tools are concurrent in their action, pleiotropic effects on target protein abundance are achievable with the simultaneous action of the diverse photoreceptor variants.