PhiReX: a programmable and red light-regulated protein expression switch for yeast

Hochrein, Lena; Machens, Fabian; Messerschmidt, Katrin; Mueller‐Roeber, Bernd

doi:10.1093/nar/gkx610

Cited by 29 publications

(49 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The first optogenetic switch developed and implemented in yeast was based on the red-light dependent interactions of the photoreceptor Phytochrome B (PhyB) and its interacting protein PIF3 from Arabidopsis thaliana (6). Ever since, this switch has been adapted in yeast with different purposes such as: reconstitution of protein activities (7), subcellular protein localization, control of cell polarity and budding phenotypes (33, 34), and reporter gene expression (YFP or GFP) (19, 35). Recently, a new red-light optogenetic switch also based on PhyB-PIF3 interaction (named PhiReX), that overcomes the obstacle of external addition of the chromophore phycocyanobilin (PCB), has been implemented and tested in yeast (35).…”

Section: Discussionmentioning

confidence: 99%

“…Ever since, this switch has been adapted in yeast with different purposes such as: reconstitution of protein activities (7), subcellular protein localization, control of cell polarity and budding phenotypes (33, 34), and reporter gene expression (YFP or GFP) (19, 35). Recently, a new red-light optogenetic switch also based on PhyB-PIF3 interaction (named PhiReX), that overcomes the obstacle of external addition of the chromophore phycocyanobilin (PCB), has been implemented and tested in yeast (35). These types of toggle switches can be quite powerful as they provide activation with a given wavelength (red) to then be turned off by another one (far-red).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

FUN-LOV: Fungal LOV domains for optogenetic control of gene expression and flocculation in yeast

Salinas

Rojas

Delgado

et al. 2018

Preprint

View full text Add to dashboard Cite

Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV; an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (Light Oxygen Voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution, and a broad dynamic range of over 1300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin encoding gene FLO1, by the FUN-LOV switch, yielded Flocculation in Light (FIL), whereas the light-controlled expression of the co-repressor TUP1 provided Flocculation in Darkness (FID). Overall, the results revealed the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV’ s ability to accurately manipulate gene expression, with a high-temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.ImportanceOptogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Herein, we report a novel optogenetic switch (FUN-LOV) based on the LOV-domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa. In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes: heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

FUN-LOV: Fungal LOV domains for optogenetic control of gene expression and flocculation in yeast

Salinas

Rojas

Delgado

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…Previous studies on TALE-based hybrids applied in human and yeast cells also reported background activity of TALE-based two-component systems in the absence of inducing signals (light or ligand; Konermann et al, 2013;Hochrein et al, 2017). This problem was circumvented by the fusion of nuclear export signals to the BC, which should prevent its nuclear import in the absence of interaction with the AC.…”

Section: N-terminal Truncations Reduce Background Activity Of the Bcmentioning

confidence: 99%

“…This problem was circumvented by the fusion of nuclear export signals to the BC, which should prevent its nuclear import in the absence of interaction with the AC. TALEreconstitution in the cytoplasm then triggers piggyback nuclear import of the BC mediated by the NLS present in the AC (Konermann et al, 2013;Hochrein et al, 2017). We followed different strategies to reduce the background activity and focused initially on the BC alone.…”

Section: N-terminal Truncations Reduce Background Activity Of the Bcmentioning

confidence: 99%

“…TEVp-site containing cyclic TALEs and cyclic transcription activator-like orthogonal repressors are cleaved in the presence of TEVp, thereby enabling access to DNA or preventing transcriptional repression, respectively (Copeland et al, 2016;Lonzarić et al, 2016). Two-component approaches are based on the separation of the binding domain (BD) from the AD and their fusion to interacting protein domains (IDs) that interact with each other in the presence of light of a specific wavelength or a specific ligand (Li et al, 2012;Konermann et al, 2013;Hochrein et al, 2017;Lo et al, 2017;Zhao et al, 2018). These systems need to be induced by a third component (light or ligand) and therefore rather represent three-component AND-gate systems.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Split-TALE: A TALE-Based Two-Component System for Synthetic Biology Applications in Planta

et al. 2019

View full text Add to dashboard Cite

Transcription activator-like effectors (TALEs) are bacterial Type-III effector proteins from phytopathogenic Xanthomonas species that act as transcription factors in plants. The modular DNA-binding domain of TALEs can be reprogrammed to target nearly any DNA sequence. Here, we designed and optimized a two-component AND-gate system for synthetic circuits in plants based on TALEs. In this system, named split-TALE (sTALE), the TALE DNA binding domain and the transcription activation domain are separated and each fused to protein interacting domains. Physical interaction of interacting domains leads to TALE-reconstitution and can be monitored by reporter gene induction. This setup was used for optimization of the sTALE scaffolds, which result in an AND-gate system with an improved signal-to-noise ratio. We also provide a toolkit of ready-to-use vectors and single modules compatible with Golden Gate cloning and MoClo syntax. In addition to its implementation in synthetic regulatory circuits, the sTALE system allows the analysis of protein-protein interactions in planta.

show abstract

The rise and shine of yeast optogenetics

Figueroa

Rojas

Romero

et al. 2020

Yeast

View full text Add to dashboard Cite

Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared with traditional chemically inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost, and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, and protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast. Take Away The budding yeast Saccharomyces cerevisiae has become a powerful biological platform for optogenetics, allowing the use of light to control diverse phenotypes. In this review, we summarized the main optogenetic tools implemented in yeast and their applications in metabolic engineering and biotechnology. Finally, we propose different sources of building blocks to expand the current repertoire of optogenetic systems in yeast.

show abstract

PhiReX: a programmable and red light-regulated protein expression switch for yeast

Cited by 29 publications

References 55 publications

FUN-LOV: Fungal LOV domains for optogenetic control of gene expression and flocculation in yeast

FUN-LOV: Fungal LOV domains for optogenetic control of gene expression and flocculation in yeast

Split-TALE: A TALE-Based Two-Component System for Synthetic Biology Applications in Planta

The rise and shine of yeast optogenetics

Contact Info

Product

Resources

About