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bioRxiv2017

The Rice XA21 Ectodomain Fused to the Arabidopsis EFR Cytoplasmic Domain Confers Resistance to Xanthomonas oryzae pv. oryzae

O_LIRice (Oryza sativa) plants expressing the XA21 cell surface receptor kinase are resistant to Xanthomonas oryzae pv.oryzae (Xoo) infection. We previously demonstrated that expressing a chimeric protein containing the EFR (ELONGATION FACTOR Tu RECEPTOR) ectodomain and the XA21 endodomain (EFR:XA21) in rice does not confer robust resistance to Xoo.\nC_LIO_LITo test if the XA21 ectodomain is required for Xoo resistance, we produced transgenic rice lines expressing a chimeric protein consisting of the XA21 ectodomain and EFR endodomain (XA21:EFR) and inoculated these lines with Xoo. We also tested if the XA21:EFR rice plants respond to a synthetic sulfated 21 amino acid derivative (RaxX21-sY) derived from the activator of XA21-mediated immunity, RaxX.\nC_LIO_LIWe found that five independently transformed XA21:EFR rice lines displayed resistance to Xoo as measured by lesion length analysis, and showed that five lines express markers of the XA21 defense response (generation of reactive oxygen species and defense response gene expression) after treatment with RaxX21-sY.\nC_LIO_LIOur results indicate that expression of the XA21:EFR chimeric receptor in rice confers resistance to Xoo. These results suggest that the endodomain of the EFR and XA21 immune receptors are interchangeable and the XA21 ectodomain is the key determinant conferring robust resistance to Xoo.\nC_LI

plant biology↗

bioRxiv2017

Variance reduction for antithetic integral control of stochastic reaction networks

The antithetic integral feedback motif recently introduced in [6] is known to ensure robust perfect adaptation for the mean dynamics of a given molecular species involved in a complex stochastic biomolecular reaction network. However, it was observed that it also leads to a higher variance in the controlled network than that obtained when using a constitutive (i.e. open-loop) control strategy. This was interpreted as the cost of the adaptation property and may be viewed as a performance deterioration for the overall controlled network. To decrease this variance and improve the performance, we propose to combine the antithetic integral feedback motif with a negative feedback strategy. Both theoretical and numerical results are obtained. The theoretical ones are based on a tailored moment closure method allowing one to obtain approximate expressions for the stationary variance for the controlled network and predict that the variance can indeed be decreased by increasing the strength of the negative feedback. Numerical results verify the accuracy of this approximation and show that the controlled species variance can indeed be decreased, sometimes below its constitutive level. Three molecular networks are considered in order to verify the wide applicability of two types of negative feedback strategies. The main conclusion is that there is a trade-off between the speed of the settling-time of the mean trajectories and the stationary variance of the controlled species; i.e. smaller variance is associated with larger settling-time.\n\nAuthor summaryHomeostasis, the ability of living organisms to regulate their internal state, is of fundamental importance for their adaptation to environmental changes and their survival. This is the reason why complex regulatory genetic networks evolved and allowed for the emergence of more and more complex organisms. Recently, the theoretical study of those regulatory networks using ideas and concepts from control theory and the design of novel ones have gained a lot of attention. Synthetic regulatory circuits are seen as elementary building blocks for the design of complex networks that need to incorporate some regulating elements to be fully functional. This is for instance the case in metabolic engineering where the production of biomolecules, such as drugs or biofuels, need to be optimized and tightly regulated. A particular circuit, the so-called antithetic integral controller, is now known to ensure homeostasis even when regulatory circuits are subject to randomness. However, it is also known that this circuit increases variability in the network. The effects of a correcting negative feedback loop on the variance are discussed here and it is shown that variability can be reduced this way. Notably, we show that there is a tradeoff between speed of the network and variability.

systems biology↗

bioRxiv2018

Beyond synthetic lethality: multiple gene interaction types play a key functional role in cancer

The phenotypic effect of perturbing a genes activity depends on the activity level of other genes, reflecting the notion that phenotypes are emergent properties of a network of functionally interacting genes. In the context of cancer, contemporary investigations have primarily focused on just one type of functional genetic interaction (GI) - synthetic lethality (SL). However, there may be additional types of GIs whose systematic identification would enrich the molecular and functional characterization of cancer. Here, we describe a novel data-driven approach called EnGIne, that applied to TCGA data identifies 71,946 GIs spanning 12 distinct types, only a small minority of which are SLs. The detected GIs explain cancer driver genes tissue-specificity and differences in patients response to drugs, and stratify breast cancer tumors into refined subtypes. These results expand the scope of cancer GIs and lay a conceptual and computational basis for future studies of additional types of GIs and their translational applications. The GI network is accessible online via a web portal [https://amagen.shinyapps.io/cancerapp/].

cancer biology↗

bioRxiv2017

A novel mitochondrial enriched antioxidant protects neurons against acute oxidative stress

Excessive reactive oxygen species (ROS) can damage proteins, lipids, and DNA, which result in cell damage and death. The outcomes can be acute, as seen in stroke, or more chronic as observed in age-related diseases such as Parkinsons disease. Here we investigate the antioxidant ability of a novel synthetic flavonoid, Proxison (7-decyl-3-hydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), using a range of in vitro and in vivo approaches. We show that, while it has radical scavenging ability on par with other flavonoids in a cell-free system, Proxison is orders of magnitude more potent than natural flavonoids at protecting neural cells against oxidative stress and is capable of rescuing damaged cells. The unique combination of a lipophilic hydrocarbon tail with a modified polyphenolic head group promotes efficient cellular uptake and mitochondrial localisation of Proxison. Importantly, in vivo administration of Proxison demonstrated effective and well tolerated neuroprotection against oxidative stress in a zebrafish model of dopaminergic neuronal loss.

cell biology↗

bioRxiv2017

ARIGAN: Synthetic Arabidopsis Plants using Generative Adversarial Network

In recent years, there has been an increasing interest in image-based plant phenotyping, applying state-of-the-art machine learning approaches to tackle challenging problems, such as leaf segmentation (a multi-instance problem) and counting. Most of these algorithms need labelled data to learn a model for the task at hand. Despite the recent release of a few plant phenotyping datasets, large annotated plant image datasets for the purpose of training deep learning algorithms are lacking. One common approach to alleviate the lack of training data is dataset augmentation. Herein, we propose an alternative solution to dataset augmentation for plant phenotyping, creating artificial images of plants using generative neural networks. We propose the Arabidopsis Rosette Image Generator (through) Adversarial Network: a deep convolutional network that is able to generate synthetic rosette-shaped plants, inspired by DC-GAN (a recent adversarial network model using convolutional layers). Specifically, we trained the network using A1, A2, and A4 of the CVPPP 2017 LCC dataset, containing Arabidopsis Thaliana plants. We show that our model is able to generate realistic 128 x 128 colour images of plants. We train our network conditioning on leaf count, such that it is possible to generate plants with a given number of leaves suitable, among others, for training regression based models. We propose a new Ax dataset of artificial plants images, obtained by our ARIGAN. We evaluate this new dataset using a state-of-the-art leaf counting algorithm, showing that the testing error is reduced when Ax is used as part of the training data.

plant biology↗

bioRxiv2015

The Arabidopsis Auxin F-box proteins AFB4 and AFB5 are Required for Response to the Synthetic Auxin Picloram

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6 (Parry et al. 2009). Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors (Dharmasiri et al. 2005; Parry et al. 2009). In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that both AFB4 and AFB5 are targets of the picloram family of auxinic herbicides. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

Plant Biology↗

bioRxiv2018

Non-adhesive alginate hydrogels support growth of pluripotent stem cell-derived intestinal organoids

Human intestinal organoids (HIOs) represent a powerful system to study human development and are promising candidates for clinical translation as drug-screening tools or engineered tissue. Experimental control and clinical use of HIOs is limited by growth in expensive and poorly defined tumor-cell-derived extracellular matrices, prompting investigation of synthetic ECM-mimetics for HIO culture. Since HIOs possess an inner epithelium and outer mesenchyme, we hypothesized that adhesive cues provided by the matrix may be dispensable for HIO culture. Here, we demonstrate that alginate, a minimally supportive hydrogel with no inherent cell adhesion properties, supports HIO growth in vitro and leads to HIO epithelial differentiation that is virtually indistinguishable from Matrigel-grown HIOs. Additionally, alginate-grown HIOs mature to a similar degree as Matrigel-grown HIOs when transplanted in vivo, both resembling human fetal intestine. This work demonstrates that purely mechanical support from a simple-to-use and inexpensive hydrogel is sufficient to promote HIO survival and development.

developmental biology↗

bioRxiv2017

A Neuropeptide Signaling System That Rapidly Enforces Paternity In The Aedes aegypti Mosquito

Female Dengue and Zika vector mosquitoes (Aedes aegypti) generally mate once, with sperm from this male fertilizing all eggs produced in her lifetime. Here we implicate HP-I, an Aedes- and male-specific neuropeptide transferred to females, and its cognate receptor in the female, NPYLR1, in rapid enforcement of paternity. HP-I mutant males were ineffective in enforcing paternity when a second male was given access to the female within 1 hour. NPYLR1 mutant females produced mixed paternity offspring at high frequency. Synthetic HP-I injected into wild-type virgins reduced successful matings, but had no effect on NPYLR1 mutant females. Asian tiger mosquito (Ae. albopictus) HP-I potently activated Ae. aegypti NPYLR1. Invasive Ae. albopictus males are known to copulate with and sterilize Ae. aegypti females, and cross-species transfer of HP-I may contribute to this phenomenon. This neuropeptide system promotes rapid paternity enforcement within Ae. aegypti, but may promote local extinction in areas where they compete with Ae. albopictus.\n\nOne Sentence SummaryAedes-specific peptide rapidly enforces paternity\n\nTextAe. aegypti females typically mate only once with one male in their lifetime, a behavior known as \"monandry\" (1). This single mating event provisions the female with sufficient sperm to fertilize the >500 eggs she will produce during her [~]4-6 week lifespan in the laboratory (2). Successful mating is capable of inducing lifetime refractoriness to subsequent insemination by other males, enforcing the paternity of the first male (3-5). In other species, males use diverse strategies to assure the paternity of their offspring, for instance physical barriers such as mating plugs found in mice (6) and Anopheline mosquitoes (7), and anti-aphrodisiac pheromones used by Drosophila melanogaster males to tag female flies as non-virgin (8). Another widely used strategy in insects is the transfer of biologically active male seminal proteins, produced by the male accessory gland and secreted into the ejaculatory duct along with sperm during insemination, to affect the sexual receptivity of the female (3, 9-13). Perhaps the best-characterized male seminal fluid protein in insects is the Drosophila fly sex peptide (11), which acts on the sex peptide receptor in the female to suppress receptivity and trigger egg production (12). Drosophila sex peptide receptor mutant females will readily remate with multiple males, and wild-type females that mate with sex peptide mutant males remain sexually receptive.

neuroscience↗

bioRxiv2017

Synthetic hormone-responsive transcription factors can monitor and re-program plant development

Developmental programs continuously sculpt plant morphology to meet environmental challenges, and these same programs have been manipulated to increase agricultural productivity1,2. Small molecule phytohormones act as signals within these programs creating chemical circuitry3 that, in many cases, has been represented in mathematical models4,5. To date, model validation and forward engineering of plant morphology has been largely restricted to adding or subtracting genes, as more nuanced tools to modulate key control parameters identified by such models in vivo are severely limited6,7. Here, we use Arabidopsis thaliana to validate a novel set of synthetic and modular hormone activated Cas9-based repressors (HACRs) that respond to three phytohormones: auxin, gibberellins and jasmonates. We demonstrate that HACRs can regulate genes in response to both exogenous hormone treatments, as well as in response to local differences in endogenous hormone levels associated with developmental events. We further show that HACRs can be used to reprogram the agriculturally relevant traits of shoot branching and phyllotaxy by tuning canalization strength, a critical control parameter predicted by mathematical models. By deploying a HACR to re-parameterize the threshold for induction of the auxin transporter PIN-FORMED1 (PIN1), we observed a decrease in shoot branching and phyllotactic noise as predicted by existing models4,5. The approach described here provides a framework for improved mapping of developmental circuitry, as well as a means to better leverage model predictions to engineer development.

plant biology↗

bioRxiv2018

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain tumour-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of sixty different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

cell biology↗

bioRxiv2018

HIV-1 Protease Evolvability is Affected by Synonymous Nucleotide Recoding

One unexplored aspect of HIV-1 genetic architecture is how codon choice influences population diversity and evolvability. Here we compared the development of HIV-1 resistance to protease inhibitors (PIs) between wild-type (WT) virus and a synthetic virus (MAX) carrying a codon-pair re-engineered protease sequence including 38 (13%) synonymous mutations. WT and MAX viruses showed indistinguishable replication in MT-4 cells or PBMCs. Both viruses were subjected to serial passages in MT-4 cells with selective pressure from the PIs atazanavir (ATV) and darunavir (DRV). After 32 successive passages, both the WT and MAX viruses developed phenotypic resistance to PIs (IC50 14.6 {+/-} 5.3 and 21.2 {+/-} 9 nM for ATV, and 5. 9 {+/-} 1.0 and 9.3 {+/-} 1.9 for DRV, respectively). Ultra-deep sequence clonal analysis revealed that both viruses harbored previously described resistance mutations to ATV and DRV. However, the WT and MAX virus proteases showed different resistance variant repertoires, with the G16E and V77I substitutions observed only in WT, and the L33F, S37P, G48L, Q58E/K, and L89I substitutions detected only in MAX. Remarkably, G48L and L89I are rarely found in vivo in PI-treated patients. The MAX virus showed significantly higher nucleotide and amino acid diversity of the propagated viruses with and without PIs (P < 0.0001), suggesting higher selective pressure for change in this recoded virus. Our results indicate that HIV-1 protease position in sequence space delineates the evolution of its mutant spectra. Nevertheless, the investigated synonymously recoded variant showed mutational robustness and evolvability similar to the WT virus.\n\nIMPORTANCELarge-scale synonymous recoding of virus genomes is a new tool for exploring various aspects of virus biology. Synonymous virus genome recoding can be used to investigate how a viruss position in sequence space defines its mutant spectrum, evolutionary trajectory, and pathogenesis. In this study, we evaluated how synonymous recoding of the human immunodeficiency virus type 1 (HIV-1) protease impacts the development of protease inhibitor (PI) resistance. HIV-1 protease is a main target of current antiretroviral therapies. Our present results demonstrate that the wild-type (WT) virus and the virus with the recoded protease exhibited different patterns of resistance mutations after PI treatment. Nevertheless, the developed PI resistance phenotype was indistinguishable between the recoded virus and the WT virus, suggesting that the synonymously recoded protease HIV-1 and the WT protease virus were equally robust and evolvable.

evolutionary biology↗

bioRxiv2018

PII1: a protein involved in starch initiation that determines granule number and size in Arabidopsis chloroplast.

The initiation of starch granule formation is still poorly understood. However, soluble starch synthase 4 (SS4) appears to be a major component of this process since it is required to synthetize the correct number of starch granules in the chloroplasts of Arabidopsis thaliana plants. A yeast-2-hybrid screen allowed the identification of several putative SS4 interacting partners. We identified the product of At4g32190 locus as a chloroplast-targeted PROTEIN INVOLVED IN STARCH INITIATION (named PII1). Arabidopsis mutants devoid of PII1 display an alteration of starch initiation process and accumulate, on average, one starch granule per plastid instead of the 5 to 7 granules found in plastids of wild-type plants. These granules are larger than in wild type and they remain flat and lenticular. pii1 mutants display wild-type growth rates and accumulate standard starch amounts. Moreover, starch characteristics, such as amylopectin chain length distribution, remain unchanged. Our results reveal the involvement of PII1 in starch priming process in Arabidopsis leaves through interaction with SS4.

plant biology↗

bioRxiv2016

In−silico studies of facilitated VEGF(s) − VEGFR(s) bindings for assessment of Lysine as an indirect Low−Mol−Wt angiogen: Experimental validation of a potential synthetic Low−Mol−Wt angiogen.

In-vivo angiogenesis process is highly conserved and is mediated through a family of peptides having VEGF-A as the lead member. A respective receptor family comprising of members VEGFR-1, 2, 3 gets expressed on the endothelial cell membrane of the vascular bed in ischemic zone along with parallel expressions of VEGF-A, B, C, D and PlGF. Degree of ischaemia is the main regulator of these coupled expressions of angiogenic peptides/factors (AFs) and respective receptor(s) for a paracrine angiogenic process to take place. Physiological angiogenesis in intrauterine growth phase is the lead process in foetal growth, organogenesis and cellular specialization. Post birth and with aging, this process gets gradually inefficient and slow. In the present in-silico study, all angiogenic factors and receptor species are examined as for their binding stability in basal unaided condition and in presence of a possible Low-Mol-Wt linkage molecule-Lysine. Also a Lysine analogue 1,6-diaminohexanoic acid has been examined for its angiogenic potential both in dry docking experiment and in cell culture assay.

Molecular Biology↗

bioRxiv2014

Expression noise facilitates the evolution of gene regulation

In studies of gene regulation, it is often tacitly assumed that the interactions between transcriptional regulators and their target promoters are finely tuned to ensure condition-appropriate gene expression of the targets. However, how natural selection might evolve such precise regulation from an initial state without regulation, is rarely discussed. In addition, the accuracy of gene regulation is affected by noise in gene expression [1]. Expression noise varies greatly across genes [2-5], suggesting that natural selection has affected noise levels, but the role of expression noise in gene regulation is currently poorly understood [6]. Here we present a combination of experimental evidence and theoretical modeling showing that the transmission of expression noise from regulators to their targets can function as a rudimentary form of gene regulation that facilitates the evolution of more finely tuned gene regulation. To assess how natural selection has affected transcriptional noise in E. coli, we evolved a large set of synthetic promoters under carefully controlled selective conditions and found, surprisingly, that native E. coli promoters show no signs of having been selected for minimizing their noise. Instead, a subset of native promoters, which are characterized by high expression plasticity and high numbers of regulatory inputs, show elevated noise levels. A general theoretical model, which recognizes that target genes are not only affected by the condition-dependent activities of their regulators, but also by the regulators noise, explains these observations. Noise transmission from regulators to their targets is favored by selection whenever regulation is imprecise, and may even constitute the main function of coupling a promoter to a regulator. Our theory provides a novel framework for understanding the evolution of gene regulation, demonstrating that in many situations expression noise is not the mere unwanted side-effect of regulatory interactions, but a beneficial function that is key to the evolvability of regulatory interactions.

Systems Biology↗

bioRxiv2017

Human Rab GTPase- And Class V Myosin-Dependent Membrane Tethering In A Chemically Defined Reconstitution System

Membrane tethering is a fundamental process essential for compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, have been reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes still remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by trans-Rab assemblies on apposing membranes. Our findings support the novel concept that Rab-family proteins provide a bona fide membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane tethering reactions.

cell biology↗

bioRxiv2017

Impairment of zebrafish reproduction upon exposure to melengestrol acetate

Synthetic progestins contamination is common in the aquatic ecosystem, which may lead to serious health problem on aquatic animals. Melengestrol acetate (MGA) has been detected in the aquatic environment; however, its potential effects on fish reproduction are largely unclear. Here, we aimed to investigate the endocrine disruption and impact of MGA on zebrafish reproduction. Six-month old reproductive zebrafish were exposed to four nominal concentrations of MGA (1,10, 100 and 200 ng/L) for 15 days. Treatment with MGA reduced the egg production with a significant decrease at 200 ng/L. The circulating concentrations of estradiol and testosterone in female zebrafish or 11-keto testosterone in male zebrafish were significantly diminished compared to the non-exposed control fish. The early embryonic development or hatching rates were unaffected during the MGA exposure. Our results indicated that MGA was a potent endocrine disruptor in fish and the fish reproduction could be impaired even during a short-term exposure to MGA.

developmental biology↗

bioRxiv2018

Model-driven design and evolution of non-trivial synthetic syntrophic pairs

Synthetic microbial communities are attractive for applied biotechnology and healthcare applications through their ability to efficiently partition complex metabolic functions. By pairing auxotrophic mutants in co-culture, nascent E. coli communities can be established where strain pairs are metabolically coupled. Intuitive synthetic communities have been demonstrated, but the full space of cross-feeding metabolites has yet to be explored. A novel algorithm, OptAux, was constructed to design 66 multi-knockout E. coli auxotrophic strains that require significant metabolite cross-feeding when paired in co-culture. Three OptAux predicted auxotrophic strains were co-cultured with an L-histidine auxotroph and validated via adaptive laboratory evolution (ALE). Time-course sequencing revealed the genetic changes employed by each strain to achieve higher community fitness and provided insights on mechanisms for sharing and adapting to the syntrophic niche. A community model of metabolism and gene expression was utilized to predict the relative community composition and fundamental characteristics of the evolved communities. This work presents a novel computational method to elucidate metabolic changes that empower community formation and thus guide the optimization of co-cultures for a desired application.

systems biology↗

bioRxiv2018

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

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.\n\nImportanceOptogenetic 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.

microbiology↗