Biology subjects

Xu, P.

Publications and source records attributed to Xu, P..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

The targeting of non-fibrillar polyQ via distinct VCP-proteasome coupling

The aggregation of proteins containing expanded poly-glutamine (polyQ) repeat sequences is a cytopathological hallmark of several dominantly inherited neurodegenerative diseases (ND), including Huntingtons disease (HD). Previously, we observed that long polyQ repeats form a fibrillar core, surrounded by amorphous and soluble intermediates, which compared to the fibrils, are more readily taken up by autophagosomes 1. In this study, we investigated how the alternative major degradation pathway in cultured cells, ubiquitin proteasome system (UPS), interacts with the different pools of polyQ. We observed that the AAA+ ATPase, valosin-containing protein VCP/p97, in collaboration with proteasomes, plays a crucial role in degrading non-fibrillar polyQ. As both VCP and proteasomes were recruited to the polyQ intermediates peripheral to the fibrils, we imaged these regions by in situ cryo-ET for subtomogram averaging. VCP predominantly adopts an ATP-bound state, often in an active processing conformation with resolved NPLOC4-like cofactor density. The region was also enriched with the 26S proteasome (26S), 20S proteasome core (20S), and 19S regulatory particles (19S). Distance analysis of the macromolecules revealed a striking proximity between VCP and the 20S in situ. This is likely mediated by VCPs C-terminal hydrophobic-tyrosine-X (HbYX) motif, which binds the 20S via hydrophobic interactions, as confirmed by cryo-EM single-particle analysis (SPA) and in vitro assays. Our study highlights a distinct VCP-20S coupling mechanism, where VCP functionally overlaps with the 19S complex in substrate unfolding, facilitating the degradation of polyQ intermediates by the 20S catalytic core.

biochemistry↗

bioRxiv2025

In Situ Detection of α-Synuclein Seeding Activity Using Quiescent Seed Amplification Assay

The prion-like propagation of proteopathic -synuclein (Syn) "seeds" is a central pathogenic mechanism in synucleinopathies. However, methods to directly visualize this "seeding activity" within intact tissue are lacking. Conventional antibody-based techniques provide only static snapshots of pathological deposits, while sensitive seed amplification assays lose spatial information via mechanical fragmentation. Here, we introduce the Syn Quiescent Seed Amplification Assay (QSAA), a method for specific, robust, in situ amplification of Syn seeds directly within tissue sections. QSAA demonstrates superior sensitivity and earlier detection than pS129 and ThS staining, revealing widespread seeding in a PD mouse model, and successfully identifies endogenous seeds in archival human brains. By integrating with immunofluorescence, IF-QSAA uncovered microglial seeding activity and established that pS129 phosphorylation and seeding activity represent distinct pathological states. This offers unprecedented spatial insights into synucleinopathy pathogenesis, holding significant promises for early diagnosis and therapeutic evaluation.

neuroscience↗

bioRxiv2025

DiNovo: high-coverage, high-confidence de novo peptide sequencing using mirror proteases and deep learning

Despite the recent advancements driven by deep learning, de novo peptide sequencing is still constrained by incomplete peptide fragmentation and insufficient protein digestion in current single protease-based proteomic experiments. Here, we present a software system, named DiNovo, for high-coverage and confidence de novo peptide sequencing by leveraging the complementarity of mirror proteases. DiNovo is empowered by several innovative algorithms, including a mirror-spectra recognition algorithm independent of pre-sequencing, two sequencing algorithms based on deep learning and graph theory, respectively, and target-decoy mapping, a method for sequencing result evaluation free of prior peptide identification. Compared with the trypsin protease used alone, DiNovo using two pairs of mirror proteases led to two to three times high-confidence amino acids sequenced. Compared with previous single-protease de novo sequencing algorithms, DiNovo achieved much higher sequence coverages. DiNovo also showed great potential as a powerful complement or alternative to database search for peptide identification with quality control.

bioinformatics↗

bioRxiv2025

STmiR: A Novel XGBoost-Based Framework for Spatially Resolved miRNA Activity Prediction

MicroRNAs (miRNAs) are critical regulators of gene expression in cancer biology; however, their spatial dynamics within tumor microenvironments (TME) remain underexplored owing to technical limitations in current spatial transcriptomics (ST) technologies. To address this gap, we present STmiR, a novel XGBoost-based framework for spatially resolved miRNA activity predictions. STmiR integrates bulk RNA-seq data (TCGA and CCLE) with spatial transcriptomics profiles to model nonlinear miRNA-mRNA interactions, achieving a high predictive accuracy (Spearmans {rho} > 0.8) across four major cancer types (breast, lung, ovarian, and prostate). Applied to 10X Visium ST datasets from nine cancers, STmiR identified six pan-cancer conserved miRNAs (e.g., hsa-miR-21, hsa-let-7a) consistently ranked in the top 40 across malignancies, and uncovered cell-type-specific regulatory networks in fibroblasts, B cells, and malignant cells. A breast cancer case study validated the utility of STmiR by linking miR-205 to androgen receptor (AR) signaling and miR-200b to epithelial-mesenchymal transition (EMT). By enabling the spatial mapping of miRNA activity, STmiR provides a transformative tool to dissect miRNA-mediated regulatory mechanisms in cancer progression and TME remodeling, with implications for biomarker discovery and precision oncology. HighlightsO_LIFirst integration of XGBoost and spatial transcriptomics: STmiR pioneers a machine learning framework to predict miRNA activity in spatially heterogeneous tissues, overcoming limitations of linear correlation-based methods. C_LIO_LIHigh accuracy and generalizability: Demonstrates robust performance (Spearmans {rho} > 0.8) across four cancer types validated by independent datasets. C_LIO_LIPan-cancer conserved miRNAs: Six miRNAs (e.g., hsa-miR-21 and hsa-let-7a) were shared across nine cancers, implicating their roles in core oncogenic pathways. C_LI Key ContributionsO_LIMethodological innovation: Combines XGBoosts nonlinear modeling with spatial transcriptomics to resolve miRNA activity in multicellular contexts. C_LIO_LIBiological discovery: Uncovers the conserved and context-dependent roles of miRNAs in tumor progression and microenvironment crosstalk. C_LIO_LITranslational impact: Provides a computational platform for identifying spatially regulated therapeutic targets in cancer. C_LI

bioinformatics↗

bioRxiv2025

Longitudinal analysis of retinal cell state transitions in RB1-deficient retinal organoids reveals the nascent cone precursors are the earliest cell-origin of human retinoblastoma

All cancers develop from malignant transformation of normal cells, while it is challenging to identify these "cells-of-origin" since it is impossible to witness the dynamic changes in human cancers. Retinoblastoma (Rb), an intraocular malignant cancer, is a typical model for study the molecular and cellular mechanisms of cancers. Although the maturing cone precursors (CPs) are proposed as the cellular origin of human Rb, it is unknow whether other retinal cells are also sensitive to RB1 inactivation. Here we developed RB1-deficient human retinal organoids (ROs) models from RB1-/- or RB1+/- human induced pluripotent stem cells (hiPSCs). RB1-/- hiPSCs developed into Rb tumors which recapitulated the features of human native Rb and had the ability to form consecutive orthotopic xenografts in mice. Importantly, RB1 loss induced the overproliferation of ATOH7+ neurogenic retinal progenitor cells (nRPCs), which disrupted the retinal development by generating proliferative, nascent retinal cells including retinal ganglion cells and CPs. scRNA-seq analysis verified the ATOH7+/RXR{gamma}+ nascent CPs survived and finally drove Rb development. In contrast, monoallelic RB1 inactivation with low pRB expression did not induce nascent CPs proliferation, but only induced nRPCs overproliferation which caused retinocytoma-like phenotype. Finally, a potential therapeutic agent for Rb was identified from multi-omics data. Our findings firstly indicate that nRPCs are the most sensitive cells to RB1 loss inducing nascent retinal cells abnormal proliferation, and ATOH7+ nascent CPs are the earliest cellular origin of human Rb, facilitating drug development for Rb.

cell biology↗

bioRxiv2025

De novo biosynthesis of cannabinoid and its analogs in Yarrowia lipolytica

Cannabis sativa has long been a cornerstone of both medicinal and cultural practices, with its therapeutic use spanning over 2,700 years. Central to its therapeutic effects are cannabinoids, which interact with the endocannabinoid system to influence various physiological processes such as anxiety, pain, and inflammation. Despite its benefits, cannabinoid production faces challenges and scarcity from plant extraction. This work leverages Yarrowia lipolytica as a platform for cannabinoid biosynthesis. By optimizing precursor supply, engineering biomolecular condensate-like dual prenyltransferase expression and expanding endogenous metabolism with a noncanonical polyketide synthase, we achieved the de novo biosynthesis of various cannabinoid and its analogs. Our engineered Y. lipolytica produced [~]3.5 mg/L of cannabigerolic acid, 18.8 mg/L of orsellinic acid, and 0.5 mg/L of cannabigerorcinic acid. Additionally, CBGA titer reached 15.7 mg/L with olivetolic acid supplement. This work demonstrates Y. lipolyticas versatility as a promising host for cannabinoid and its analogs production, which opens avenues for further research and medicinal applications.

synthetic biology↗

bioRxiv2025

Dynamic Monomer-Dimer Transition in Ligand-induced Apelin Receptor Activation

G-protein-coupled receptors (GPCRs) are significant signal transducers that exist as monomers and in multiple oligomeric forms. However, molecular mechanism driving their dynamic interconversion to regulate intricate signaling in class A GPCRs remains elusive, compounding our understanding of their related pathophysiological functions. Here, we present a set of 12 assemblies of the apelin receptor (APLNR), including dimeric apo state, agonistic small molecule- or nanobody-bound state of monomeric and dimeric APLNR with and without G-proteins, providing a detailed dynamic view of the monomer-dimer transition. High-resolution cryo-EM structures reveal that different ligands induce varying degrees of pre-dissociation of dimers in the absence of G-protein, with G-protein coupling facilitating the transition from dimeric to monomeric receptor. Functional studies further highlight the critical role of cholesterol clusters in stabilizing the APLNR dimers. These insights enhance our understanding of the dynamic regulation of class A GPCRs across different aggregated forms and advance the rational drug design strategies aimed at selectively modulating of APLNR signaling.

molecular biology↗

bioRxiv2025

Dynamic early recruitment of GAK-Hsc70 regulates coated pit maturation

Clathrin-mediated endocytosis (CME) is the process by which clathrin assembles on the plasma membrane to form clathrin-coated pits (CCPs), which then invaginate, accumulate cargo and are released by fission from the membrane to form clathrin-coated vesicles (CCVs). A transition of nascent CCPs from flat-to-curved has been observed by various methods. However, what drives this transition remains unknown and controversial. GAK and its chaperone protein, Hsc70, are well-known to mediate clathrin release from CCVs and several studies have observed a late burst of GAK recruitment as CCVs form. Other studies have proposed that early recruitment of GAK-Hsc70 could function to provide the necessary energy source to remodel nascent flat clathrin lattices, replacing hexagons with pentagons and enabling a gain of curvature and invagination of the growing CCP; however, direct functional evidence is lacking. Here we show that GAK knockdown inhibits CCP formation and invagination. Furthermore, mutations in the J domain of GAK that abolish Hsc70 recruitment to and activation at CCPs, lead to the accumulation of GAK at CCPs, hinder CCP stabilization and invagination and result in a striking increase in the proportion of short-lived, abortive CCPs. These findings support the hypothesis that GAK-Hsc70 promotes the turnover and remodeling of nascent clathrin assemblies required for curvature development during CME. Significance StatementGAK and its chaperone protein, Hsc70, are known to be recruited to clathrin-coated vesicles (CCVs) to mediate clathrin uncoating. Previous studies have proposed that early recruitment of GAK-Hsc70 to CCPs could function to remodel nascent flat clathrin lattices, replacing hexagons with pentagons and enabling a gain of curvature of the assembling polymeric coat. However, there are conflicting views, and direct functional evidence is lacking. Here we show that GAK knockdown inhibits CCP formation and invagination. A detailed domain-specific mutational analysis of GAK pinpointed the importance of J domain-Hsc70 interactions in regulating crucial early steps of CME including CCP stabilization and invagination. These findings support a hypothesis that GAK-Hsc70 promotes turnover of clathrin at nascent CCPs required for curvature development.

cell biology↗

bioRxiv2025

Making plant tissue accessible for cryo-electron tomography

Cryo-Electron Tomography (cryo-ET) allows to visualize the molecular architecture of pristinely preserved cells and tissues. The workflow of sample preparation for cryo-ET is rather complex; it involves vitrification by rapid freezing followed by cryo-Focused Ion Beam (FIB) milling rendering the volumes of interest thin enough for cryo-ET data acquisition. The established protocols for single cells grown on or deposited on EM-grids are not suitable for multicellular plant tissues. Plunge-freezing does not yield vitrified samples in most cases and must be replaced by high-pressure freezing. This, in turn, necessitates extensive modifications of the subsequent FIB milling procedures. In this communication we describe procedures for sample screening, targeted FIB milling guided by cryo-fluorescence microscopy and a novel lamella trimming step that allows to obtain homogenously thin lamellae suitable for cryo-ET. We have tested all the steps along the workflow with a variety of plant tissues including the moss Physcomitrium patens and tissues of Arabidopsis thaliana and Limonium bicolor. We could demonstrate that the workflow optimized for plant tissues allows to attain subnanometer resolution in cases where subtomogram averaging is applicable.

plant biology↗

bioRxiv2025

AMPK- and cGAS-Mediated Dual Sensing of Glucose and dsDNA Synergizes to Enhance Antitumor Immunity

Intratumoral glucose availability is linked considerably to tumor growth, immune evasion, and metastasis. However, the precise molecular mechanisms by which glucose levels govern cellular and immune responses against tumors remain incompletely understood. Here, we explored the context of tumors of intersections between AMP-activated protein kinase (AMPK) and cGAS-STING signaling, a key pathway initiating antitumor immunity. We unexpectedly found that AMPK activation robustly enhances cGAS-STING signaling within cancer and immune cells, which potently impacts melanoma and colorectal cell senescence, organoid apoptosis, and the infiltration of CD4+ and CD8+ T lymphocytes. Using classic or newly developed AMPK agonist Aldometanib, TBK1S511E/S511E knock-in (KI) mice, glucose metabolic modulator, as well as AMPK1/2 dKO melanoma, we demonstrated that dual sensing of intratumoral glucose and dsDNA integrated by the AMPK-TBK1 cascade is essential for initiating antitumor immunity and suppressing cancer progressions. Notably, novel AMPK agonists or intervening in glucose glycolysis by Lonidamine synergized effectively with STING agonists, substantially inhibiting melanoma growth. Therefore, these findings unravel a concise mechanism integrating glucose metabolism and innate DNA sensing into cancer cell fate and propose an effective therapeutic strategy to enhance antitumor immunity. HIGHLIGHTSO_LIDual and synergistic sensing of glucose and dsDNA facilitates antitumor immunity in melanoma and colorectal cancer; C_LIO_LIAMPK substitutes intratumoral cGAS-STING-IRF3 signaling through TBK1 phosphorylation at S511; C_LIO_LIGlucose deficiency induces cancer cell senescence, apoptosis, and T lymphocyte infiltration via the AMPK-TBK1 cascade; C_LIO_LINewly developed AMPK agonists synergize with STING agonists to retrieve striking tumor suppression. C_LI

cancer biology↗

bioRxiv2025

Evaluating the toxicity of methanol, formaldehyde and formate on the growth fitness of Yarrowia lipolytica

Liquid C1 feedstock, including methanol, formaldehyde and formic acid can be easily manufactured from greenhouse gases CO2 and CH4. Yarrowia lipolytica, isolated from marine ecosystem, is a non-conventional yeast which is capable of assimilating multiple range of hydrophobic compounds including alcohols, lipids, hydrocarbons, and volatile fatty acids et al. This yeast has been extensively engineered to produce lipids, carotenoids, squalene and other natural products. Herein we tested the growth fitness of Y. lipolytica when exposed to various one-carbon (C1) liquid feedstocks (methanol, formaldehyde, and formic acid). We firstly captured the cell growth kinetics with an automatic Cell Growth Analyzer and fitted the data with a Monod-type growth model. We obtained the inhibition constants of the three liquid C1s from the growth fitness dose-response curves. We found that the lagging phase duration is exponentially correlated with the toxicity of the tested C1 solvents. Notably, Y. lipolytica exhibited tolerance to methanol (IC50 871 mM) and formic acid (IC50 42.6 mM), but showed sensitivity to formaldehyde (IC50 3.8 mM). We next chose fromate as the co-substrate to cultivate Y. lipolytica, confirmed that Y. lipolytica can upcycle formic acid to improve lipid yields and biomass in fed-batch culture. This work broadens Y. lipolyticas substrate scope and facilitates sustainable and cost-efficient production of fuels and chemicals.

synthetic biology↗

bioRxiv2025

Multiple interactions mediate the localization of BLTP2 at ER-PM contacts to control plasma membrane dynamics

BLTP2/KIAA0100, a bridge-like lipid transfer protein, was reported to localize at contacts of the endoplasmic reticulum (ER) with either the plasma membrane (PM) or recycling tubular endosomes depending on the cell type. Our findings suggest that mediating bulk lipid transport between the ER and the PM is a key function of this protein as BLTP2 tethers the ER to tubular endosomes only after they become continuous with the PM and that it also tethers the ER to macropinosomes in the process of fusing with the PM. We further identify interactions underlying binding of BLTP2 to the PM, including phosphoinositides, the adaptor proteins FAM102A and FAM102B, and also N-BAR domain proteins at membrane-connected tubules. The absence of BLTP2 results in the accumulation of intracellular vacuoles, many of which are connected to the plasma membrane, pointing to a role of the lipid transport function of BLTP2 in the control of PM dynamics.

cell biology↗

bioRxiv2025

Genomic Insights into the Demographic History and Local Adaptation of Wild Boars Across Eurasia

Wild boars exhibit genetic and phenotypic diversity shaped by migrations and local adaptations. Its expansion across Eurasia, especially in central Asia, remains underexplored. Here, we present newly sequenced whole-genome data of 47 wild boars from Eastern Asia, Central Asia, and Europe, combined with 49 existing genomes, creating a comprehensive dataset of 96 individuals. Our analyses show that Asian wild boars and Southeast Asian Suids split [~]3.6 Mya, with Central Asian and Southern Chinese ancestors diverging [~]1.8 Mya. The split between Central Asian and European-Near East ancestors occurred [~]0.9 Mya, followed by a European-Near East divergence [~]0.6 Mya. We identify signatures of local adaptation in Central Asian populations, including two positively selected variants in LPIN1, associated with cold adaptation, and a missense mutation in ALPK2, linked to meat traits. These findings provide insights into wild boar dispersal and adaptation, and shed light on domestic pigs breeding.

evolutionary biology↗

bioRxiv2025

Engineering low pH-tolerant Komagataella phaffii for industrial-ready itaconic acid production via methanol biorefinery

Methanol biorefinery stands as a cornerstone strategy in the pursuit of sustainable biochemical production from non-food feedstocks. Despite its vast potential, bioconverting methanol into high titer biochemical production remains a formidable challenge. In this study, we have successfully engineered Komagataella phaffii to efficiently produce itaconic acid (IA), one of the top 12 building block chemicals, utilizing methanol as the sole carbon source. Our approach involved strategic metabolic engineering interventions--such as enhancing the supply of cis-aconitate and citrate precursors, minimizing isocitrate diversion, facilitating efficient formaldehyde assimilation, and optimizing IA export mechanisms--culminating in an unprecedented IA titer of 103.8 g/L in a 5-L bioreactor through fed-batch fermentation at pH 5.5. Remarkably, even at a reduced pH of 3.5, the strain still yielded 50.4 g/L of IA, highlighting the industrial viability of methanol biorefinery. This systematic work lays a robust foundation for high-titer production via methanol biorefinery, offering valuable insights for future research and applications.

synthetic biology↗

bioRxiv2025

Bayesian phylodynamic inference of population dynamics with dormancy

Many organisms employ reversible dormancy, or seedbank, in response to environmental fluctuations. This life-history strategy alters fundamental eco-evolutionary forces, leading to distinct patterns of genetic diversity. Two models of dormancy have been proposed based on the average duration of dormancy relative to coalescent timescales: weak seedbank, induced by scheduled seasonality (e.g., plants, invertebrates), and strong seedbank, where individuals stochastically switch between active and dormant states (e.g., bacteria, fungi). The weak seedbank coalescent is statistically equivalent to the Kingman coalescent with a scaled mutation rate, allowing the use of existing inference methods. In contrast, the strong seedbank coalescent differs fundamentally, as only active lineages can coalesce, while dormant lineages cannot. Additionally, dormant individuals typically mutate at a slower rate than active ones. Consequently, despite the significant role of dormancy in the eco-evolutionary dynamics of many organisms, no methods currently exist for inferring population dynamics involving dormancy and associated parameters. We present a Bayesian framework for jointly inferring a latent genealogy, seedbank parameters, and evolutionary parameters from molecular sequence data under the strong seedbank coalescent. We derive the exact probability density of genealogies sampled under the strong seedbank coalescent, characterize the corresponding likelihood function, and present efficient computational algorithms for its evaluation based on our theoretical framework. We develop a tailored Markov chain Monte Carlo sampler and implement our inference framework as a package SeedbankTree within BEAST2. Our work provides both a theoretical foundation and practical inference framework for studying the population genetic and genealogical impacts of dormancy.

evolutionary biology↗

bioRxiv2025

Clinical and Taxonomic Characterization of a Novel Urinary Tract Pathogen Providencia lanzhouensis

BackgroundThe genus Providencia includes species of ecological and clinical significance, with some acting as opportunistic pathogens in hospital-acquired infections such as urinary tract infection (UTI). However, overlapping phenotypic traits and genetic similarities pose challenges for accurate species characterization. This study exemplifies this challenge by identifying a novel Providencia isolate through genomic analysis. ResultsIn this study, strain PAZ2 was isolated from the urine sample of a hospitalized patient in Lanzhou, Gansu Province, China. Initial mass spectrometry analysis identified the strain as Providencia alcalifaciens. It exhibits a resistance profile against antibiotics including ampicillin, tetracycline, tigecycline, polymyxin B, colistin, trimethoprim-sulfamethoxazole, and ciprofloxacin. However, whole-genome sequence analysis indicated that this strain is a novel species with an average nucleotide identity (ANI) values ranging from 79.92% to 94.73% and digital DNA-DNA hybridization (dDDH) values between 21.2% and 57.6% when compared with all known Providencia species. Furthermore, strain PAZ2 exhibited unique biochemical properties that differentiated it from all previously described species within the genus. ConclusionsGenomic and biochemical evidence supports PAZ2 as a novel Providencia species, which we named Providencia lanzhouensis sp. nov. This finding enhances the taxonomy of Providencia and underscores its clinical relevance, notably in UTIs and other nosocomial infections. Accurate identification is crucial for guiding effective treatment and preventing misdiagnosis. Further studies should investigate its pathogenicity, antimicrobial resistance, and epidemiology to inform infection control measures.

microbiology↗

bioRxiv2024

PlantRing: A high-throughput wearable sensor system for decoding plant growth, water relations and innovating irrigation

The combination of flexible electronics and plant science has generated various plant-wearable sensors, yet challenges persist in their applications in real-world agriculture, particularly in high-throughput settings. Overcoming the trade-off between sensing sensitivity and range, adapting them to a wide range of crop types, and bridging the gap between sensor measurements and biological understandings remain the primary obstacles. Here we introduce PlantRing, an innovative, nano-flexible sensing system designed to address the aforementioned challenges. PlantRing employs bio-sourced carbonized silk georgette as the strain sensing material, offering exceptional resolution (tensile deformation: < 100 m), stretchability (tensile strain up to 100 %), and remarkable durability (season long), exceeding existing plant strain sensors. PlantRing effectively monitors plant growth and water status, by measuring organ circumference dynamics, performing reliably under harsh conditions and being adaptable to a wide range of plants. Applying PlantRing to study fruit cracking in tomato and watermelon reveals novel hydraulic mechanism, characterized by genotype-specific excess sap flow within the plant to fruiting branches. Its high-throughput application enabled large-scale quantification of stomatal sensitivity to soil drought, a traditionally difficult-to-phenotype trait, facilitating drought tolerant germplasm selection. Combing PlantRing with soybean mutant led to the discovery of a potential novel function of the GmLNK2 circadian clock gene in stomatal regulation. More practically, integrating PlantRing into feedback irrigation achieves simultaneous water conservation and quality improvement, signifying a paradigm shift from experience- or environment-based to plant-based feedback control. Collectively, PlantRing represents a groundbreaking tool ready to revolutionize botanical studies, agriculture, and forestry.

plant biology↗

bioRxiv2024

General assessment of genetically-encoded reporters in Yarrowia lipolytica

The development of genetically-encoded reporters is important to characterize gene expression dynamics and investigate cellular-level events. Unlike Bakers yeast, the thick cell wall and the high oil content in oleaginous yeast have restricted our ability to establish efficient fluorescence or enzyme-based reporters. In this book chapter, we detailed the protocol for how to clone and construct fluorescence reporters including hrGFP, TurboGFP, and mScarlet-I3, and the Nanoluc luciferase reporters. We quantified the fluorescence/luciferase reporter efficiency with 96-well microplate readers, flow cytometry, and confocal microscopy. Our results indicate that TurboGFP and hrGFP have a relatively low signal/noise ratio, and mScarlet-I3 yields a very high signal/noise ratio. Compared to fluorescence, luciferase Nanoluc exhibits the highest signal/noise ratio. The downside of using luciferase is the relatively laborious procedure and the related high cost. This chapter may guide us to establish an efficient and reliable reporter system to study gene expression or protein-labeling in nonconventional yeast.

bioengineering↗