Curated Optogenetic Publication Database

Abstract: Methylotrophic yeasts such as Pichia pastoris are widely used for heterologous protein production because they contain strong and tightly regulated promoters. However, the use of methanol as an inducer presents several practical challenges, including toxicity, flammability, high oxygen demand during fermentation, and increased production costs. To overcome these limitations, researchers have been working on redesigning the AOX1 regulatory system and developing alternative induction strategies that do not rely on methanol. One promising approach is optogenetics, which uses light to control gene expression in a non-invasive way. These systems rely on light-sensitive proteins such as phytochromes, cryptochromes, LOV-domain proteins, and UVR8, allowing gene activity to be regulated in a precise and reversible manner without adding chemical inducers to the culture medium. This review brings together key advances in yeast optogenetics, with a focus on the EL222 system, highlighting its implementation for light-controlled heterologous protein production in P. pastoris and its broad application in synthetic biology and metabolic engineering in Saccharomyces cerevisiae. The growing versatility and scalability of EL222-based circuits highlight their potential to reshape both fundamental research and industrial bioprocessing through safer, more controllable, and energy-efficient gene regulation strategies.

Abstract: The BcWCL1 protein is a blue-light photoreceptor from the fungus Botrytis cinerea. This protein has a central role in B. cinerea circadian regulation and is an ortholog to WC-1 from Neurospora crassa. The BcWCL1 and WC-1 proteins have similar protein domains, including a LOV (Light Oxygen Voltage) domain for light sensing, two PAS (Per Arnt Sim) domains for protein-protein interaction, and a DNA binding domain from the GATA family. Recently, the blue-light response of BcWCL1 was demonstrated in a version without PAS domains (BcWCL1PAS∆). Here, we demonstrated that BcWCL1PAS∆ is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1PAS∆ enables transcriptional activation as a single component in yeast. By using chimeric transcription factors and the luciferase reporter gene, we assessed the transcriptional activity of different fragments of the N-terminal and C-terminal regions of BcWCL1PAS∆, identifying a functional transcriptional activation domain (AD) in the N-terminal region that belongs to the 9aaTAD family. Finally, we determined that the transcriptional activation levels of BcWCL1PAS∆ AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1PAS∆ protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics.

Abstract: Botrytis cinerea possesses a complex light-sensing system composed of eleven photoreceptors. In B. cinerea, bcwcl1 encodes for the BcWCL1 protein, the orthologue of the blue-light photoreceptor WC-1 from Neurospora crassa. The functional partner of BcWCL1 is the BcWCL2 protein, both interacting in the nucleus and forming the B. cinerea white collar complex (BcWCC). This complex is required for photomorphogenesis and circadian regulation. However, no molecular evidence shows a light-dependent interaction between the BcWCC components or light-sensing capabilities in BcWCL1. In this work, by employing a yeast two-hybrid system that allows for the in vivo analysis of protein-protein interactions, we confirm that BcWCL1 and BcWCL2 interact in the absence of light as well as upon blue-light stimulation, primarily through their PAS (Per-Arnt-Sim) domains. Deletion of the PAS domains present in BcWCL1 (BcWCL1PAS∆) or BcWCL2 (BcWCL2PAS∆) severely impairs the interaction between these proteins. Interestingly, the BcWCL1PAS∆ protein shows a blue-light response and interacts with BcWCL2 or BcWCL2PAS∆ upon light stimulation. Finally, we demonstrate that BcWCL1 and BcWCL1PAS∆ respond to blue light by introducing a point mutation in the photoactive cysteine, confirming that both proteins are capable of light sensing. Altogether, the results revealed the complexity of protein-protein interactions occurring between the core elements of the B. cinerea circadian clock.