Control of cellular events by optogenetic tools is a powerful approach to manipulate cellular functions in a minimally invasive manner. A common problem posed by the application of optogenetic tools ...is to tune the activity range to be physiologically relevant. Here, we characterized a photoreceptor of the light–oxygen–voltage (LOV) domain family of Phaeodactylum tricornutum aureochrome 1a (AuLOV) as a tool for increasing protein stability under blue light conditions in budding yeast. Structural studies of AuLOVwt, the variants AuLOVM254, and AuLOVW349 revealed alternative dimer association modes for the dark state, which differ from previously reported AuLOV dark-state structures. Rational design of AuLOV-dimer interface mutations resulted in an optimized optogenetic tool that we fused to the photoactivatable adenylyl cyclase from Beggiatoa sp. This synergistic light-regulation approach using two photoreceptors resulted in an optimized, photoactivatable adenylyl cyclase with a cyclic adenosine monophosphate production activity that matches the physiological range of Saccharomyces cerevisiae. Overall, we enlarged the optogenetic toolbox for yeast and demonstrated the importance of fine-tuning the optogenetic tool activity for successful application in cells.
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•Light-induced protein stabilization.•Alternative dark state dimer structures of Phaeodactylum tricornutum aureochrome 1a.•Rational engineering of AuLOV dimerization.•Photoactivatable adenylyl cyclase from Beggiatoa sp. engineered for budding yeast.•Physiological relevant regulation of cAMP production in Saccharomyces cerevisae.
Optogenetics has great potential for biotechnology and metabolic engineering due to the cost‐effective control of cellular activities. The usage of optogenetics techniques for the biosynthesis of ...bioactive molecules ensures reduced costs and enhanced regulatory possibilities. This requires development of efficient methods for light‐delivery during a production process in a fermenter. Here, we benchmarked the fermenter production of a low‐caloric sweetener in Saccharomyces cerevisiae with optogenetic tools against the production in small scale cell culture flasks. An expression system based on the light‐controlled interaction between Cry2 and Cib1 was used for sweet‐protein production. Optimization of the fermenter process was achieved by increasing the light‐flux during the production phase to circumvent shading by yeast cells at high densities. Maximal amounts of the sweet‐protein were produced in a pre‐stationary growth phase, whereas at later stages, a decay in protein abundance was observable. Our investigation showcases the upscaling of an optogenetic production process from small flasks to a bioreactor. Optogenetic‐controlled production in a fermenter is highly cost‐effective due to the cheap inducer and therefore a viable alternative to chemicals for a process that requires an induction step.
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Optogenetics has great potential for biotechnology and metabolic engineering due to the cost‐effective control of cellular activities. In this paper the authors benchmarked the fermenter production of a low‐caloric sweetener in Saccharomyces cerevisiae with optogenetic tools against the production in small scale cell culture flasks. The paper results show that optogenetic regulation strategies might be used efficiently in biotechnology applications.
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.
•Optogenetic cell cycle control in a eukaryotic production strain.•Proteome-wide analysis of optogenetically triggered reallocation of cellular resources.•Detection of affected metabolic subsystems by protein-constrained flux modelling.•Demonstration of generically optimized production processes by optogenetics in yeast.
Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane ...pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.
