in-vivo-monitoring-transcriptional-activity-during-metabolic

<em>In Vivo</em> Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

ANID-Millennium Science Initiative-Millennium Institute for Integrative Biology (iBio)

Pontificia Universidad Católica de Chile

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.

The rise and shine of yeast optogenetics.

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In , optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus . We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Modular and Molecular Optimization of a LOV (Light-Oxygen-Voltage)-Based Optogenetic Switch in Yeast.

 possesses a complex light-sensing system composed of eleven photoreceptors. In ,  encodes for the BcWCL1 protein, the orthologue of the blue-light photoreceptor WC-1 from . The functional partner of BcWCL1 is the BcWCL2 protein, both interacting in the nucleus and forming the  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  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 (BcWCL1) or BcWCL2 (BcWCL2) severely impairs the interaction between these proteins. Interestingly, the BcWCL1 protein shows a blue-light response and interacts with BcWCL2 or BcWCL2 upon light stimulation. Finally, we demonstrate that BcWCL1 and BcWCL1 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  circadian clock.

Interactions between Core Elements of the  Circadian Clock Are Modulated by Light and Different Protein Domains.

For comprehensive gene expression analyses of the phytopathogenic fungus , which infects a number of plant taxa and is a cause of substantial agricultural losses worldwide, we developed BEB, a web-based  gene Expression Browser. This computationally inexpensive web-based application and its associated database contain manually curated RNA-Seq data for . BEB enables expression analyses of genes of interest under different culture conditions by providing publication-ready heatmaps depicting transcript levels, without requiring advanced computational skills. BEB also provides details of each experiment and user-defined gene expression clustering and visualization options. If needed, tables of gene expression values can be downloaded for further exploration, including, for instance, the determination of differentially expressed genes. The BEB implementation is based on open-source computational technologies that can be deployed for other organisms. In this case, the new implementation will be limited only by the number of transcriptomic experiments that are incorporated into the platform. To demonstrate the usability and value of BEB, we analyzed gene expression patterns across different conditions, with a focus on secondary metabolite gene clusters, chromosome-wide gene expression, previously described virulence factors, and reference genes, providing the first comprehensive expression overview of these groups of genes in this relevant fungal phytopathogen. We expect this tool to be broadly useful in  research, providing a basis for comparative transcriptomics and candidate gene identification for functional assays.

The  Gene Expression Browser.

 is a necrotrophic fungus characterized mainly by its wide host range of infected plants. The deletion of the  gene (), which encodes for a blue-light receptor/transcription factor, causes a decrease in virulence, particularly when assays are conducted in the presence of light or photocycles. However, despite ample characterization, the extent of the light-modulated transcriptional responses regulated by BcWCL1 remains unknown. In this study, pathogen and pathogen:host RNA-seq analyses, conducted during non-infective in vitro plate growth and when infecting  leaves, respectively, informed on the global gene expression patterns after a 60 min light pulse on the wild-type B05.10 or ∆ strains. The results revealed a complex fungal photobiology, where the mutant did not react to the light pulse during its interaction with the plant. Indeed, when infecting , no photoreceptor-encoding genes were upregulated upon the light pulse in the ∆ mutant. Differentially expressed genes (DEGs) in  under non-infecting conditions were predominantly related to decreased energy production in response to the light pulse. In contrast, DEGs during infection significantly differ in the B05.10 strain and the ∆ mutant. Upon illumination at 24 h post-infection in planta, a decrease in the  virulence-associated transcripts was observed. Accordingly, after a light pulse, biological functions associated with plant defense appear enriched among light-repressed genes in fungus-infected plants. Taken together, our results show the main transcriptomic differences between wild-type  B05.10 and ∆ after a 60 min light pulse when growing saprophytically on a Petri dish and necrotrophically over .

Genome-Wide Characterization of Light-Regulated Gene Expression in  Reveals Underlying Complex Photobiology.

Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a "lights-on timer" logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks.

Luis F. Larrondo

ANID-Millennium Science Initiative-Millennium Institute for Integrative Biology (iBio)

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast