Biology subjects

Whiteway, M.

Publications and source records attributed to Whiteway, M..

14 recordsLinked to original sources

bioRxiv2026

Heterologous engineering of receptors using OrthoRep (HERO) for directed evolution of GPCRs in yeast

Engineering G protein-coupled receptors (GPCRs) for biosensing applications remains challenging due to structural and functional constraints when such proteins are expressed in a heterologous host. OrthoRep offers the continuous accumulation and selection of signal-enhancing mutations in yeast, yet it has not been applied to GPCR evolution. We apply OrthoRep-driven mutagenesis to evolve opioid GPCRs and improve yeast-based biosensors: first, we express the human -opioid receptor (OPRM1) on the OrthoRep p1 plasmid and couple ligand activation to growth. Serial cell passaging of the biosensor generated functionally diverse mutants. However, mutations that decouple receptor activation from selection arose over longer passaging campaigns, halting evolution. We resolve this issue using a site-specific recombinase that re-introduces evolved variants onto the p1 landing pad of an unmutated biosensor, allowing continued selection. This led to an ~18-fold improvement in sensitivity of a mutant OPRM1 over wt-OPRM1. Additional polymerases were added to the platform, broadening the mutational profiles available. Assembling these components forms the platform we call HERO (Heterologous Engineering of Receptors using OrthoRep). Lastly, we deploy HERO using automation to improve the human {delta}-opioid receptor's weak response to an agonist by ~40-fold. Sensitivity also improved for a structurally dissimilar ligand assayed on the same evolved variants.

synthetic biology↗

bioRxiv2025

Body temperature drives azole tolerance in Candida albicans by hindering the autophagic degradation of Erg11

Human body temperature has been shown to limit the antifungal efficacy of azoles; however, the precise mechanisms underlying this limitation remain insufficiently understood. In this study, we observed that short-term exposure (24 h) to human body temperature (37{square}{degrees}C), in contrast to 30{square}{degrees}C, significantly enhances the tolerance of Candida albicans to azoles without inducing resistance. Our findings suggest that this increased tolerance is due to a reduction in the degradation of C. albicans Erg11 at 37{square}{degrees}C compared to 30{square}{degrees}C, thereby promoting azole tolerance. This phenomenon occurs because Erg11 degradation is mediated by autophagy, which is inhibited in C. albicans at 37{square}{degrees}C. The suppression of autophagy at this temperature may be associated with elevated mitochondrial production of reactive oxygen species, leading to mitochondrial dysfunction. In response to heat stress-induced mitochondrial dysfunction, autophagy-related proteins such as Atg8 accumulate around the mitochondria, while their levels in the endoplasmic reticulum decrease, consequently inhibiting autophagy in C. albicans. These findings suggest that promoting the degradation of Erg11 in C. albicans may serve as a viable strategy to augment the fungicidal efficacy of azoles in the human host.

microbiology↗

bioRxiv2025

Tri-Functional CRISPR Screen Reveals Overexpression of QDR2 and QDR3 Transporters Increase Fumaric Acid Production in Kluyveromyces marxianus

Organic acids such as fumaric acid are widely used in the food and beverage industry as acidulants and preservatives, while also serving as versatile precursors for industrially relevant compounds. Fumaric acid is still predominantly produced through petroleum-derived processes. To enhance production efficiency and diversify supply, we are engineering Kluyveromyces marxianus as a biosynthetic platform from renewable feedstocks. In previous work, we have established K. marxianus Y-1190 as a host for lactose valorization based on its high growth rate on lactose and its tolerance for acid conditions. Here, we establish a trifunctional genome-wide library for K. marxianus using CRISPR activation, interference, and deletion to allow identification of gene expression perturbations that enhance tolerance to fumaric acid. We determined that deletion of ATP7, encoding a subunit of the mitochondrial F1F0 ATP synthase, and overexpression of QDR2 and QDR3, two previously uncharacterized members of the 12-spanner H antiporter (DHA1) family in K. marxianus, can enhance fumaric acid tolerance. We also found that integrated overexpression of both QDR2 and QDR3 in a {Delta}FUM1 background strain improved titers of fumaric acid production from 0.26 g L-1 to 2.16 g L-1. Together, these results highlight roles for membrane transport and mitochondrial function in enabling fumaric acid tolerance and production in K. marxianus. HIGHLIGHTS- Trifunctional CRISPR-AID enables gene activation, interference & deletion in K. marxianus. - Genome-wide CRISPR-AID screen identifies guides conferring fumaric acid tolerance. - verexpressing of QDR2 and QDR3 increases fumaric acid tolerance and production. - Deletion of mitochondrial gene ATP7 significantly improves fumaric acid tolerance. - Fermentation of QDR2 and QDR3 overexpression strain yields 2.16 g L-{superscript 1} fumaric acid.

synthetic biology↗

bioRxiv2025

Characterization of ORF19.7608 (PPP1), a Biofilm-induced Gene of Candida albicans

The opportunistic human pathogen Candida albicans is an important cause of nosocomial infections, in large part because of its propensity to form biofilms on indwelling medical devices such as catheters. The formation of these biofilms is controlled by a complex transcriptional network and involves over a thousand genes, many of which are uncharacterized. We have investigated three genes (ORF19.4654, ORF19.7608, and PBR1), found only in C. albicans and closely related species, that are highly induced under biofilm conditions and encode small proteins with N-terminal signal sequences. Through the construction of fluorescent protein fusions, we have examined the location of the encoded proteins in both planktonic and biofilm cells. Orf19.4654-Scarlet and Pbr1-Scarlet were localized to the vacuole under both conditions. In contrast, the Orf19.7608-GFP fusion generated a punctate pattern only under biofilm conditions and was designated Ppp1 (Punctate Pattern Protein 1). The Ppp1-GFP puncta were similar in location, stability, and size to those formed by the eisosome subunit Sur7, but co-localization studies suggest that Ppp1 and Sur7 define separate elements. The PPP1 mutation does not cause a distinct phenotype under various stress conditions or in the presence of antifungals and does not impact biofilm formation and biomass. These data suggest that while the expression and cellular localization of Ppp1 appear controlled by conditions generating biofilms, and define a unique subcellular localization pattern, Ppp1 protein function is not essential for biofilm formation. IMPORTANCEBiofilm formation is a virulence factor of medical importance in C. albicans. Identifying the biological function and cellular localization of biofilm-related proteins can help in their characterization and understanding of the biofilm network. Microscopy was employed to identify the subcellular localization of putative biofilm proteins, aiming to provide insight into their possible functions. Prb1 and Orf19.4654 localized to the vacuole, while Orf19.7608 (Ppp1) formed puncta. Phenotypic assays to investigate the uniqueness of PPP1 revealed that it does not play a major role in the stress response pathways or antifungal activity. Overall, our study provides insight into the localization of the products of Candida-specific biofilm genes.

microbiology↗

bioRxiv2025

Sequencing of a dairy isolate unlocks Kluyveromyces marxianus as a host for lactose valorization

The use of genetically modified non-conventional yeast provides significant potential for the bioeconomy by diversifying the tools available for the development of sustainable and novel products. In this study, we sequenced and annotated the genome of Kluyveromyces marxianus Y-1190 to establish it as a platform for lactose valorization. The strain was chosen for rapid growth on lactose-rich dairy permeate, high transformation efficiency, and ease of culturing in bioreactors. Genomic sequencing revealed that K. marxianus Y-1190 possesses single nucleotide polymorphisms associated with efficient lactose metabolism. The strain is diploid with notable genomic heterogeneity, which appears to be critical for its robust growth and acid tolerance. To further exploit this platform strain, we developed protocols for gene and chromosome manipulation using CRISPR editing, constructed and validated a series of promoters compatible with MoClo vectors, and designed synthetically inducible promoters for K. marxianus. These tools enable precise control over gene expression, allowing for the tailored optimization of metabolic pathways and production processes. The synthetic promoters provide flexibility for dynamic expression tuning, while the CRISPR-based editing protocols facilitate targeted genetic modifications with high efficiency. Together, these advancements significantly enhance the genetic toolbox for K. marxianus, positioning it as a versatile platform for industrial biotechnology. These tools open new opportunities for the sustainable production of bio-based chemicals, fuels, and high-value products, leveraging lactose-rich feedstocks to contribute to a circular economy.

synthetic biology↗

bioRxiv2023

Screening non-conventional yeasts for organic acid tolerance and engineering Pichia occidentalis for production of cis,cis-muconic acid

Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organisms prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L-1 adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis,cis-muconic acid (CCM), a precursor to AA. By developing a genome editing toolkit for P. occidentalis, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L-1), yield (0.134 g g-1 glucose) and productivity (0.511 g L-1 h-1) that surpasses all metrics achieved using S. cerevisiae. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.

synthetic biology↗

bioRxiv2023

The Adr1 transcription factor directs regulation of the ergosterol pathway and azole resistance in C. albicans

Transcription factors play key roles in cellular regulation and are critical in the control of drug resistance in the fungal pathogen Candida albicans. We found that activation of the transcription factor C4_02500C_A (Adr1) conferred significant resistance against fluconazole. In Saccharomyces cerevisiae, Adr1 is a carbon-source-responsive zinc-finger transcription factor required for transcription of the glucose-repressed gene ADH1 and of genes required for ethanol, glycerol, and fatty acid utilization. Motif scanning of promoter elements suggests that Adr1 may be rewired in fungi and governs the ergosterol synthesis pathway in C. albicans. Because previous studies have identified the zinc-cluster transcription factor Upc2 as a regulator of the ergosterol pathway in both fungi, we examined the relationship of Adr1 and Upc2 in sterol biosynthesis in C. albicans. Phenotypic profiles of either ADR1 and UPC2 modulation in C. albicans showed differential growth in the presence of fluconazole; either adr1 or upc2 homozygous deletion results in sensitivity to the drug while their activation generates a fluconazole resistant strain. The rewiring from ergosterol synthesis to fatty acid metabolism involved all members of the Adr1 regulon except the alcohol dehydrogenase Adh1, which remains under Adr1 control in both circuits and may have been driven by the lifestyle of S. cerevisiae, which requires the ability to both tolerate and process high concentrations of ethanol.

genetics↗

bioRxiv2022

Candida albicans exhibits heterogeneous and adaptive cytoprotective responses to anti-fungal compounds

Candida albicans is an opportunistic human pathogen which represents a significant threat to human health and is associated with substantial socio-economic burden. Current antifungal treatments fail at least in part because C. albicans can initiate a strong drug tolerance response, allowing cells to grow at concentrations above their minimal inhibitory concentration. Our goal is to better characterize this cytoprotective tolerance program at the molecular single cell level. We present here a nano-liter droplet-based fungal single cell transcriptomics platform capable of profiling thousands of individual C. albicans SC5314 cells in an efficient manner. Profiles of untreated cells partition into three transcriptional clusters with each highlighting a cell cycle checkpoint coupled with specific metabolic and stress responses, as perhaps expected. After just two days post-treatment with fluconazole, surviving cells bifurcate into two distinct subpopulations: the so-called response involving upregulation of protein translation, rRNA processing and mitochondrial cellular respiration, and the {beta} response involving processes and stress responses that assist damaged cells. By extending our time series to six days and profiling with other antifungals and bioactive compounds, we provide evidence that surviving cells transition from the to {beta} responses mediated by the Ribosome Assembly Stress Response (RASTR).

microbiology↗

bioRxiv2021

The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 toolkit with three components: (i) a set of optimized S. pyogenes Cas9-guide RNA (gRNA) expression vectors with five selectable markers and the option to either pre-clone or co-transform the gRNAs; (ii) vectors for the one-step construction of integration cassettes expressing an untagged or GFP/RFP/HA-tagged gene of interest at one of three levels, supporting localization and overexpression studies; and (iii) integration cassettes containing moderately expressed GFP- or RFP-tagged compartmental markers for colocalization experiments. These components allow rapid, high efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. To demonstrate the ease of use, we applied our complete set of compartmental markers to co-label all target subcellular compartments with GFP and RFP. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle to simplify yeast research.

synthetic biology↗

bioRxiv2021

A deep learning approach to capture the essence of Candida albicans morphologies

We present deep learning-based approaches for exploring the complex array of morphologies exhibited by the opportunistic human pathogen C. albicans. Our system entitled Candescence automatically detects C. albicans cells from Differential Image Contrast microscopy, and labels each detected cell with one of nine vegetative, mating-competent or filamentous morphologies. The software is based upon a fully convolutional one-stage object detector and exploits a novel cumulative curriculum-based learning strategy that stratifies our images by difficulty from simple vegetative forms to more complex filamentous architectures. Candescence achieves very good performance on this difficult learning set which has substantial intermixing between the predicted classes. To capture the essence of each C. albicans morphology, we develop models using generative adversarial networks and identify subcomponents of the latent space which control technical variables, developmental trajectories or morphological switches. We envision Candescence as a community meeting point for quantitative explorations of C. albicans morphology.

microbiology↗

bioRxiv2021

Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols

The yeast Saccharomyces cerevisiae is a powerful tool for studying G protein-coupled receptors (GPCRs) as they can be functionally coupled to its pheromone response pathway. However, some exogenous GPCRs, including the mu opioid receptor, are non-functional in yeast, which may be due to the presence of the fungal sterol ergosterol instead of the animal sterol cholesterol. We engineered yeast to produce cholesterol and introduced the human mu opioid receptor, creating an opioid biosensor capable of detecting the peptide DAMGO at an EC50 of 62 nM and the opiate morphine at an EC50 of 882 nM. Furthermore, introducing mu, delta, and kappa opioid receptors from diverse vertebrates consistently yielded active opioid biosensors that both recapitulated expected agonist binding profiles with EC50s as low as 2.5 nM and were inhibited by the antagonist naltrexone. Additionally, clinically relevant human mu opioid receptor alleles, or variants with terminal mutations, resulted in biosensors that largely displayed the expected changes in activity. We also tested mu opioid receptor-based biosensors with systematically adjusted biosynthetic intermediates of cholesterol, enabling us to relate sterol profiles with biosensor sensitivity. Finally, cholesterol-producing and sterol intermediate biosensor backgrounds were applied to other human GPCRs, resulting in SSTR5, 5-HTR4, FPR1 and NPY1R signaling with varying degrees of cholesterol dependence. Our sterol-optimized platform will be a valuable tool in generating human GPCR-based biosensors, aiding in ongoing receptor deorphanization efforts, and providing a framework for high-throughput screening of receptors and effectors.

synthetic biology↗

bioRxiv2020

Candida albicans exhibits distinct cytoprotective responses to anti-fungal drugs that facilitate the evolution of drug resistance

We developed a modified protocol for nanolitre droplet-based single cell sequencing appropriate for fungal settings, and used it to transcriptionally profiled several thousands cells from a prototrophic Candida albicans population and several drug exposed colonies (incl. fluconazole, caspofungin and nystatin). Thousands of cells from each colony were profiled both at early and late time points post-treatment in order to infer survival trajectories from initial drug tolerance to drug resistance. We find that prototrophic C. albicans populations differentially and stochastically express cytoprotective epigenetic programs. For all drugs, there is evidence that tolerant individuals partition into distinct subpopulations, each with a unique survival strategy involving different regulatory programs. These responses are weakly related to changes in morphology (shift from white to opaque forms, or shift from yeast to filamentous forms). In turn, those subpopulations that successfully reach resistance each have a distinct multivariate epigenetic response that coordinates the expression of efflux pumps, chaperones, transport mechanisms, and cell wall maintenance. Live cell fluorescent imaging was used to validate predictions of which molecular responses most often led to survival after drug exposure. Together our findings provide evidence that C. albicans has a robust toolkit of short-term epigenetic cytoprotective responses designed to "buy time" and increase the chance of acquiring long-term resistance.

genomics↗

bioRxiv2020

The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans

Zinc cluster transcription factors are essential fungal specific regulators of gene expression. In the dimorphic pathogen Candida albicans, they control processes ranging from metabolism and stress adaptation to mating, virulence, and antifungal resistance. Here, we have identified the gene CaORF19.1604 as encoding a zinc cluster transcription factor that acts as a regulator of filament development. Hyperactivation of CaORF19.1604, which we have named RHA1 for Regulator of Hyphal Activity, leads to a wrinkled colony morphology under non-hyphal growth conditions, to pseudohyphal growth and filament formation, to invasiveness and enhanced biofilm formation. Cells with activated Rha1 are sensitive to cell wall modifying agents such as Congo red and the echinocandin drug caspofungin but show normal sensitivity to fluconazole. RNA-sequencing-based transcriptional profiling of the activated Rha1 strain reveals the up-regulation of genes for core filamentation and cell-wall-adhesion-related proteins such as Als1, Als3, Ece1, and Hwp1. Upregulation is also seen for the genes for the hyphal-inducing transcription factors Brg1 and Ume6 and genes encoding several enzymes involved in arginine metabolism, while downregulation is seen for the hyphal repressor Nrg1. The deletion of BRG1 blocks the filamentation caused by activated Rha1, while null mutants of UME6 result in a partial block. Deletion of RHA1 can partially reduce healthy hyphal development triggered by environmental conditions such as Spider medium or serum at 37{degrees}C. In contrast to the limited effect of either single mutant, the double rha1 ume6 deletion strain is totally defective in both serum and Spider medium stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization in even brg1 ume6 double mutants. Our results thus suggest that in response to external signals, Rha1 functions to facilitate the switch from an Nrg1 controlled yeast state to a Brg1/Ume6 regulated hyphal state. Author SummaryCandida albicans is the predominant human fungal pathogen, generating a mortality rate of 40% in systemically infected patients. The ability of Candida albicans to change its morphology is a determinant of its tissue penetration and invasion in response to variant host-related stimuli. The regulatory mechanism for filamentation includes a complex network of transcription factors that play roles in regulating hyphae associated genes. We identify here a new regulator of filamentation from the zinc cluster transcription factor family. We present evidence suggesting that this transcription factor assists the Nrg1/Brg1 switch regulating hyphal development.

microbiology↗

bioRxiv2018

Behavioral response to visual motion impacts population coding in the mouse visual thalamus

Visual motion is a ubiquitous component of animals sensory experience and its encoding is critical for navigation and movement. Yet its impact on behavior and neural coding is not well understood. Combining pupillometry with cellular calcium imaging measurements of thalamocortical axons in awake behaving mice, we examined the impact of arousal and behavioral state on encoding of visual motion in the visual thalamus. We discovered that back-to-front visual motions elicits a robust behavioral response that shapes tunings of visual thalamic responses. Consistent with an arousal mechanism, the effects were pronounced during stillness and weak or absent during locomotor activity and under anesthesia. The impact on neuronal tuning was specific, biasing population response patterns in favor of back-to-front motion. The potent influence of visual motion on behavioral state dynamically affect sensory coding at early visual processing stages. Further research is required to reveal the circuitry and function of this novel mechanism.

neuroscience↗