Biology subjects

Zhong, L.

Publications and source records attributed to Zhong, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Gymnosperm FLS2 orthologues mediate conserved flg22 perception and divergent responses to flg22 variants

O_LIWhether gymnosperm FLAGELLIN-SENSING 2 (FLS2) orthologues are functional receptors and whether their flg22-recognition spectra have already diversified remain unclear, despite the central role of FLS2 in flagellin perception in angiosperms. C_LIO_LIHere, we identified two gymnosperm FLS2 orthologues, GbFLS2 from Ginkgo biloba and PtFLS2 from Pinus tabuliformis, and analysed their function using transient and stable expression in the Nicotiana benthamiana fls2 mutant, in planta cross-linking assays, AlphaFold3 modelling and structure-guided mutagenesis. C_LIO_LIBoth receptors restored flg22Pst-triggered reactive oxygen species production and MAPK activation, physically associated with flg22Pst, and required conserved residues for flg22Pst recognition. In stable transgenic plants, both receptors mediated flg22Pst-triggered PTI outputs and flg22Pst-induced resistance to Pseudomonas syringae. PtFLS2 additionally mediated responsiveness to flg22Rso and enhanced resistance to Ralstonia solanacearum, whereas GbFLS2 retained a typical flg22Pst-recognition profile. C_LIO_LIThese findings provide direct evidence that gymnosperm FLS2 orthologues can function as bona fide flagellin receptors in a heterologous angiosperm background, and further suggest that diversification in flg22 recognition had already emerged within gymnosperms. C_LI

plant biology↗

bioRxiv2026

A CRISPR-Cas9 screen Reveals STEEP1 as a Key Host Dependency Factor for Epstein-Barr Virus Latent Membrane Protein 1 Trafficking and Signaling

The Epstein-Barr virus (EBV) oncogene Latent membrane protein 1 (LMP1) is essential for B-cell transformation into continuously growing lymphoblastoid cell lines. LMP1 traffics to plasma membrane and intracellular signaling sites to mimic aspects of signaling by the B cell co-receptor CD40. LMP1 is expressed in many EBV-associated cancers, including post-transplant lymphoma, Hodgkin lymphoma, T/NK lymphoma and nasopharyngeal carcinoma, where it activates key growth and survival pathways. LMP1 signaling is also implicated in multiple sclerosis pathogenesis. To identify host dependency factors that support LMP1 trafficking and signaling, we performed a human genome-wide CRISPR-Cas9 screen in B cells. The screen identified both known and previously uncharacterized mediators of LMP1 signaling. The ER resident protein STEEP1, implicated in DNA sensor STING trafficking and signaling, was a top screen hit. Importantly, STEEP1 did not score in our prior B cell CRISPR screen for factors that support CD40 signaling, suggesting specificity. STEEP1 depletion strongly impaired LMP1 signaling, including activation of NF-kB and MAP kinase pathways. Mechanistically, STEEP1 associated with LMP1 in a manner dependent on the N-terminal cytoplasmic tail and supported LMP1 egress from the ER to signaling sites in both B and epithelial cells. Collectively, these findings reveal STEEP1 as a key host factor that supports trafficking of newly synthesized LMP1 molecules to intracellular signaling sites and highlights LMP1/STEEP1 interaction as a novel therapeutic target. ImportanceEpstein-Barr virus (EBV) infects most people worldwide. While infection is often benign, it causes infectious mononucleosis, is associated with a range of lymphomas, nasopharyngeal and gastric carcinoma and is a major trigger for autoimmune disease, including multiple sclerosis. The EBV encoded oncogene LMP1 is a key driver of EBV pathogenesis, and its signaling is necessary for viral immortalization of B lymphocytes into continuously growing lymphoblasts (LCLs). Here, we performed a CRISPR genetic screen to identify host factors that support continuous, ligand-independent signaling by LMP1. This analysis identified an ER-resident protein called STEEP1, previously implicated in support of trafficking of the DNA sensor STING, as a key LMP1 partner. We found that STEEP1 associates with LMP1 and supports LMP1 trafficking out of the endoplasmic reticulum to cellular signaling sites. As STEEP1 knockout impaired LMP1 function and LCL survival, our study identifies the STEEP1/LMP1 complex as a therapeutic target.

microbiology↗

bioRxiv2026

scDEcrypter: Uncertainty-aware differential expression analysis for viral infection in scRNA-seq

Single-cell RNA-seq studies of viral infection are limited by sparse viral reads, under-labeled infected cells, and bystander responses that confound differential expression (DE) analysis. We introduce scDEcrypter, a penalized two-way mixture model that leverages partial labels for infection status and additional variables such as cell type. Our approach employs data-splitting to avoid double-dipping and enables fast, likelihood-based inference for DE analysis. Through simulations and applications on two different viral infection datasets, scDE-crypter demonstrated improved recovery of infected cell states and identified more biologically coherent infection-associated genes and enriched pathways.

genomics↗

bioRxiv2026

T2T Genome and Population Resequencing Reveal OfCCD4 Alleles Orchestrate Petal Color and Scent in Osmanthus fragrans

Osmanthus fragrans is prized for its floral color and fragrance, both key targets for genetic improvement. However, the lack of a complete genome assembly and comprehensive population structure hinders gene dissection and marker development for breeding. To investigate the genetic basis of color-scent interaction, a telomere-to-telomere (T2T) genome assembly is generated, and whole-genome resequencing of 100 cultivars is performed. Integrative population and metabolomic analyses reveal a trade-off: orange-red ( Aurantiacus) cultivars accumulate high /{beta}-carotene but low aromatic apocarotenoids (/{beta}-ionone), while yellow-white cultivars show the opposite pattern. Divergence mapping identifies OfCCD4 as the major underlying locus. Three alleles are characterized--functional (A), partially functional (aDel), and a frameshift null (aStop)--with aStop strictly co-segregating with the Aurantiacus phenotype. Transient and stable transformations confirm that A and aDel cleave /{beta}-carotene into /{beta}-ionone, while aStop abolishes this activity. A co-dominant PCR marker is developed based on allele-specific polymorphisms. OfCCD4 is thus established as the key regulator of the color-scent trade-off in osmanthus. The identified alleles and molecular marker enable rapid, low-cost genotyping at the seedling stage--offering particular value for marker-assisted selection in ornamental breeding, where extended juvenility makes phenotypic selection highly time- and resource-intensive.

plant biology↗

bioRxiv2026

Systemic CYP3A inhibition by ritonavir enables selective targeting of hypoxic tumour cells by prodrugs of DNA-PK inhibitors

Hypoxic tumour cells are resistant to many forms of cancer therapy, particularly radiotherapy. Hypoxia-activated prodrugs (HAPs) can potentially address this problem through selective release of drugs ( effectors) in oxygen-deficient microenvironments, via metabolic reduction of a nitro(hetero)aromatic trigger moiety. While many such HAPs show marked selectivity for hypoxia in cell culture, none have yet been approved for clinical use. Here, we report HAPs that release a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK) which, like hypoxia, is a major contributor to radioresistance. These ether-linked HAPs provide hypoxia-dependent radiosensitisation in cell culture, but in mice systemic generation of the DNA-PK inhibitor is observed. Using in vitro hepatic metabolism models we demonstrate hypoxia-independent metabolic activation of HAP 4 via oxidation of its linker, which is mediated exclusively by CYP3A. We extend this finding to HAPs with other triggers, linkers and effectors. The clinically used CYP3A-specific inhibitor ritonavir suppressed hepatic metabolism of 4 under oxia without interfering with its hypoxia-dependent activation. In mice, ritonavir markedly enhanced oral bioavailability of the HAP, suppressed systemic formation of the DNA-PK inhibitor, and selectively radiosensitised HCT116 tumours but not the gastrointestinal tract in the radiation field. This combination offers the prospect of increasing the therapeutic ratio of DNA-PK inhibitor-mediated radiosensitisation in patients.

pharmacology and toxicology↗

bioRxiv2026

Artificial Recombination of Nonribosomal Peptide Synthetases Rapidly Evolves Natural Products

Efficient engineering of nonribosomal peptide synthetases (NRPSs) is a key strategy for expanding valuable peptide natural products. Current bioinformatics-guided approaches are often constrained by a set of predefined fusion sites, remaining experimentally challenging in most NRPS systems. Here we present the Recombineering Accelerated Evolution (RACE), which harnesses Red/ET recombineering mediated partially matched homologous recombination to recapitulate recombination-driven NRPS evolution on a highly accelerated timescale. Application of RACE to six known NRPS gene clusters generated 830 recombinants and yielded over 600 novel peptides including novel bis-lipopeptides. These recombinants reveal 112 previously unrecognized recombination fusion sites, providing an extensive landscape for NRPS evolution and more useful resources for guiding NRPS engineering. The RACE establishes a new paradigm for programmable NRPS evolution and enables rapid discovery of bioactive peptides.

synthetic biology↗

bioRxiv2026

Orofacial behaviors, not eye movements, govern neural activity in mouse visual cortex

Neural activity in mouse primary visual cortex (V1) correlates strongly with orofacial movements. Such strong modulation has not been found in the primate visual cortex during eye fixation [1], which led to the suggestion that the modulation may primarily depend on eye movements in both species [2]. Here we examined the influence of eye movements on neural activity in mouse visual cortex both in complete darkness and in the presence of different types of visual input. In all cases, we found that eye movements explain a smaller fraction of neural activity variance compared to orofacial behaviors. Additionally, we found that eye movements were correlated to orofacial movements, such as whisking and sniffing, and thus may be indirectly correlated to neural activity. These results further emphasize the impact of movement signals on mouse visual cortex during free viewing behavior.

neuroscience↗

bioRxiv2026

Proteo-transcriptomics and morphometrics of teleost cardiac cells define regulatory networks and exercise-induced cardiomyocyte hypertrophy and hyperplasia

Zebrafish and medaka are powerful cardiovascular models, yet cellular and molecular investigations of adult heart cells have been constrained by suboptimal dissociation and characterization methods. To overcome these barriers, we developed a physiological-temperature workflow that generates high-yield, viable single-cell suspensions for FACS, imaging, and low-input molecular profiling. Using transgenic fluorescent reporters, we consistently isolate [~]6,000 cardiomyocytes per adult zebrafish ventricle and [~]12,000 from medaka, preserving cellular, structural, and molecular integrity. Single-cell morphometrics revealed cardiomyocyte heterogeneity and demonstrated that swimming exercise induces both hypertrophy and hyperplasia, while ventricular injury triggers expansion of regenerative gata4 cardiomyocytes. We integrated proteomics and RNA-seq from FACS-purified cells to construct cell-type-specific proteo-transcriptomic atlases. Functional enrichment, transcription factor, and network analyses identified protein hubs and regulatory circuits defining cardiomyocyte and endothelial cell identity. Our platform delivers cell-type-resolved molecular datasets of adult teleost cardiac cells, establishing a systems-level resource for heart regeneration research, cardiovascular disease modeling, and drug discovery. TeaserA robust workflow for isolation, FACS, imaging, and multi-omics profiling of cardiomyocytes and cardiac endothelial cells in fish. HIGHLIGHTSO_LITemperature-optimized dissociation and standardized FACS enable high-yield isolation of viable cardiac cells. C_LIO_LISingle-cell imaging uncovers morphological heterogeneity in adult ventricular cardiomyocytes. C_LIO_LISustained exercise induces both hypertrophy and hyperplasia of zebrafish cardiomyocytes. C_LIO_LIProteo-transcriptomics defines core molecular programs and interaction networks in cardiomyocytes and endothelial cells. C_LI

systems biology↗

bioRxiv2026

Healthcare Infrastructure Shapes Evolutionary Trade-offs and Geographic Dissemination of Multidrug-Resistant Acinetobacter baumannii

Antimicrobial-resistant pathogens pose an existential threat to modern medicine, yet the evolutionary forces driving their adaptation in healthcare systems remain largely unexplored. We revealed that hospital network architecture functions as a primary selective pressure, driving pathogen evolution through infrastructure-dependent virulence-transmission trade-offs. Phylogenomic analysis of 5,023 Acinetobacter baumannii isolates across Chinas centralized healthcare system identifies two co-existing evolutionary strategies: a virulence-optimized clade (ESL2.4) that spread slowly (20.4 km per year) in low-connectivity hospitals, and a transmission-optimized clade (ESL2.5) that disseminate rapidly (65.2 km per year) through mega-city healthcare hubs, likely attributed to its capsule conversion and increased upper respiratory colonization. Comparative analysis with European A. baumannii populations demonstrates that healthcare connectivity, not geography, governs pathogen distribution through convergent genomic adaptations. Our simulation suggests competitive asymmetries of the two clades, following the ecotype principle and enabling stable coexistence. The COVID-19 pandemic provided a natural experiment validating these mechanisms: outpatient restrictions reduced transmission-optimized lineage spread by 89%, while virulence-optimized lineage persisted through inpatient networks. These findings establish healthcare infrastructure as a critical evolutionary driver with immediate implications for predicting and controlling antimicrobial resistance emergence across diverse healthcare systems.

microbiology↗

bioRxiv2026

Lamin A/C directs nucleosome-scale chromatin remodeling to define early lineage segregation in mammals

Chromatin organization underlies gene regulation and cell fate specification, yet how nucleosome-scale chromatin structure contributes to lineage segregation during early development remains unknown. Here, we resolved chromatin ultrastructure during the first lineage decision in mouse and human, which forms the pluripotent inner cell mass (ICM) and trophectoderm (TE). To achieve this, we developed dual-tilt chromatin electron tomography (2T-ChromEMT) that allows multiscale visualization of chromatin architecture. Our analysis reveals that TE cells of both species display denser chromatin with nucleosome aggregation at the nuclear periphery. We show upregulation of the nuclear matrix protein Lamin A/C within the TE lineage across mouse, human, and opossum embryos, indicating that its regulatory role is conserved across eutherian and marsupial species. Loss of Lamin A/C reduces heterochromatin at the nuclear lamina in TE cells, reactivates pluripotency genes, and impairs mouse blastocyst expansion and human blastoid formation. These findings define the nucleosome-resolution chromatin signatures of early mammalian lineages and establish Lamin A/C-mediated chromatin organization as a conserved mechanism in the exit from pluripotency and maintenance of trophectoderm identity.

developmental biology↗

bioRxiv2025

Evolutionary landscape of oral microbiome over 100,000 years

The human oral microbiome represents an intimate and enduring partnership between host and microbial communities, yet its evolutionary trajectory across deep time remains largely uncovered. Here we reconstruct the global history of the oral microbiome over 102,400 years using a global collection of 1854 oral samples spanning 61 countries. We reveal that major cultural transitions of the Neolithic Revolution, industrialization, and recent medical advances have driven a directional, globally coordinated co-evolution of oral microbial ecology, function, and population structure. Network analysis identifies two universal microbial modules of dental calculus modules: the commensal-enriched DCM1 module and the pathogen-enriched DCM2, both exhibiting progressive ecological polarization. DCM2 surged around 4,000 years ago with agricultural intensification but subsequently declined in modern European populations while persisting at high levels in African and some American groups, a geographic divide mirroring socioeconomic inequities. Species-level analyses demonstrate widespread directional changes, with obligate anaerobes declining as aerotolerant taxa expand, reflecting a carbohydrate-associated functional remodeling toward starch metabolism. Phylogenomic reconstruction reveals contrasting evolutionary trajectories. Pauljensenia mediterranea experienced near-extinction following a severe bottleneck, while Actinomyces israelii underwent a selective sweep with rapid population expansion 1,000 years ago. Critically, the oral resistome has accelerated in the antibiotic era, resulting in [~]33-50-fold surge of highly mobile, clinically relevant antimicrobial resistance genes in the last century, with Streptococcus acting as a central hub of dissemination. Collectively, these results position the oral microbiome as an active participant that has co-evolved with human subsistence, urbanization and medical practice, with direct implications for global oral health, antimicrobial resistance and interventions aimed at restoring resilient microbial states.

microbiology↗

bioRxiv2025

Unraveling the Molecular and Physiological Roles of Signal Peptide Peptidase A (SppA) in Flavobacterium columnare

Columnaris disease, caused by Flavobacterium columnare, represents one of the most economically devastating bacterial infections in global freshwater aquaculture. Despite its significant impact, the molecular mechanisms underlying F. columnare pathogenesis remain largely unexplored due to the challenge in targeted genomic manipulation. Signal peptide peptidase A (SppA) plays a crucial role in bacterial protein secretion by degrading residual signal peptides after protein translocation, yet its function in F. columnare physiology and virulence has not been characterized. Here, we employed a targeted gene deletion approach to investigate the role of sppA in F. columnare. The {Delta}sppA mutant exhibited pleiotropic phenotypes including increased outer membrane vesicle (OMV) production (3.8-fold higher compared to the wild type), reduced biofilm formation, and loss of gliding motility. Transcriptomic analysis of the {Delta}sppA mutant revealed significant upregulation of genes involved in membrane stress response and efflux pump system, including algU, osmC and the genes in the MacAB-TolC efflux system, compared to the wild-type state. Importantly, the artificial infection experiment demonstrated the mutants significantly attenuated virulence in freshwater Medaka (Oryzias latipes), with a 20% higher survival rate of fish compared to the wild type. Our findings reveal that SppA is essential for maintaining membrane homeostasis in F. columnare and serves as one of the virulence factors during columnaris infection. These results provide important insights into the biological function of the sppA gene in F. columnare and highlight the complex relationship between bacterial protein secretion, membrane integrity, and pathogenesis. ImportanceF. columnare causes significant economic loss in freshwater aquaculture. Understanding the molecular mechanisms underlying F. columnare pathogenesis is crucial for developing new ways for disease control. Our findings reveal that SppA is essential for gliding motility, adhesion, biofilm formation and maintaining membrane homeostasis in F. columnare, which serves as one of the virulence factors during columnaris infection. In addition, outer membrane vesicles (OMVs) and MacA/MacB/TolC tripartite efflux pump served as a compensatory mechanism for enhanced peptide metabolites secretion to manage the accumulation of misfolded proteins resulting from the sppA deficiency. These results provide important insights into the biological function of the sppA gene in F. columnare and highlight the complex relationship between bacterial protein secretion, membrane integrity, and pathogenesis.

genetics↗

bioRxiv2025

Epstein-Barr Virus Latent Membrane Protein 1 targets cIAP1, cIAP2 and TRAF2 for Proteasomal Degradation to Activate the Non-canonical NF-κB Pathway

The Epstein-Barr virus oncoprotein Latent Membrane Protein 1 (LMP1) is expressed in multiple malignancies and is critical for B-cell immortalization. LMP1 constitutively activates NF-{kappa}B signaling pathways, which are essential for EBV-mediated B cell transformation and for transformed B cell survival. Reverse genetic analysis revealed two LMP1 regions critical for primary human B cell immortalization, termed transformation effector site (TES) 1 and 2, which activate multiple host growth and survival pathways, in particular NF-{kappa}B. Of these, only TES1 signaling is required for B-cell transformation within the first several weeks of infection. TES1 signaling is also critical for EBV-transformed lymphoblastoid B-cell survival. However, precisely how TES1 initiates NF-{kappa}B signaling has remained incompletely understood. Here, we provide multiple lines of evidence that TES1 associates with cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2) in a tumor necrosis factor associated factor 3 (TRAF3) dependent manner. TES1 signaling drives cIAP1 autoubiquitination and targets TRAF2, cIAP1 and 2 for proteasomal degradation in a TRAF3 dependent manner. Overexpression of either cIAP1 or 2 impaired LMP1 TES1-mediated non-canonical NF-{kappa}B activation. Collectively, these studies suggest that LMP1 TES1 initiates non-canonical NF-{kappa}B signaling distinctly from CD40 and other host immunoreceptors, thereby highlighting a therapeutic target.

microbiology↗

bioRxiv2025

Gene-Corrected Basal Cells Restore CFTR In Vitro; Transplants Regenerate Epithelium in a Preclinical Sinus Model

BackgroundCystic fibrosis (CF) is caused by mutations in the CFTR gene, leading to epithelial dysfunction and progressive lung disease. Although CFTR modulators have transformed care, [~]10% of people with CF remain without effective therapy. Durable, mutation-agnostic approaches are urgently needed. MethodWe used a lentiviral (LV) vector to deliver wild-type CFTR to airway basal cells derived from 13 paediatric CF participants with a range of genotypes. Transduced cells were assessed for transgene expression, epithelial differentiation, and CFTR function using air-liquid interface (ALI) cultures. Separately, to evaluate regenerative capacity in vivo, LVGFP-transduced rabbit airway basal cells were transplanted into the denuded nasal septum of healthy New Zealand white rabbits using a biocompatible scaffold. ResultsTransduced basal cells retained multilineage differentiation capacity, forming well-organized, pseudostratified epithelium with intact barrier function and ciliary activity. CFTR channel activity was restored to levels comparable to or exceeding those achieved with elexacaftor/tezacaftor/ivacaftor (ETI), including in individuals with nonsense mutations. Combined CFTR transduction plus ETI treatment showed additive benefit. In vivo, transplanted rabbit basal cells engrafted and differentiated to regenerate a mucociliary epithelium, with improved nasal potential difference and mucociliary clearance compared to scaffold-only controls. ConclusionOur study demonstrates that LV-mediated CFTR gene addition restores CFTR function in vitro across genotypes and supports epithelial regeneration in a clinically relevant animal airway model. This two-part platform offers a scalable path toward cell therapies for all people with CF and may have broader applications in upper airway epithelial repair.

cell biology↗

bioRxiv2025

Secondary structure transitions and dual PIP2 binding define cardiac KCNQ1-KCNE1 channel gating

The KCNQ1+KCNE1 potassium channel complex forms the slow delayed rectifier current (IKs) critical for cardiac repolarization. Loss-of-function variants in KCNQ1 and KCNE1 cause long QT syndrome types 1 and 5 (LQT1/LQT5), accounting for over one-third of clinical LQTS cases. Despite prior structural work on KCNQ1 and KCNQ1+KCNE3, the structural basis of KCNQ1+KCNE1 remains unresolved. Using cryo-EM and electrophysiology, we determined high-resolution (2.5-3.4 [A]) structures of human KCNQ1+KCNE1 in both closed and open states. KCNE1 occupies a pivotal position at the interface of three KCNQ1 subunits, inducing seven "helix-to-loop" transitions in KCNQ1 transmembrane segments. These structural rearrangements: 1) stabilize the closed pore and the conformation of the intermediate voltage-sensing domain, thereby determining channel gating, ion permeation, and single channel conductance; 2) enable a dual-PIP2 modulation mechanism, where one PIP2 occupies the canonical site, while the second PIP2 bridges the S4-S5 linker, KCNE1, and the adjacent S6, stabilizing channel opening; 3) create a fenestration capable of binding compounds specific for KCNQ1+KCNE1 (e.g., AC-1). Together, these findings reveal a previously unrecognized large-scale secondary structural transition during ion channel gating that fine-tunes IKs function and provides a foundation for targeted LQTS therapy development.

biophysics↗

bioRxiv2025

Dendritic computation for rule-based flexible categorization

A hallmark of intelligent behavior is the ability to flexibly respond to external sensory inputs based on dynamically changing rules. A central question is how neurons in the brain implement computations underlying intelligent behaviors. The neocortical pyramidal neurons use their elaborated dendritic arbors to segregate a plethora of inputs and dynamically integrate them--a process known as dendritic computation--which may play important roles in rule-dependent sensory processing. However, evidence directly linking dendritic computation with intelligent cognitive behaviors has been absent. Here we combine two-photon imaging and a rule-switching flexible categorization task in mice to show that a projectome-defined extratelencephalic (ET) cortical layer 5 (L5) neurons in the auditory cortex integrate dendritic rule information and somatic sensory input to enable rule-dependent flexible categorization. The apical dendrite and soma within the same ET neurons exhibit distinct compartmental representations for sensory and rule information, with the soma predominantly encoding sensory information and the dendrites representing inferred task rule. Simultaneous optogenetic dendritic inhibition and two-photon imaging revealed that dendritic rule coding is essential for somatic output of flexible categorization. Our findings indicate that nonlinear dendritic integration of rule and sensory information constitutes a neuronal computational mechanism underlying rule-switching flexible decision-making.

neuroscience↗

bioRxiv2025

Changing the initiation unit of nonribosomal peptide synthetases to access underexplored biosynthetic potential

Nonribosomal peptide synthases (NRPSs) are large multimodular enzymes, capable of synthesizing nonribosomal peptides (NRPs) with diverse structures and bioactivities. Genome sequencing revealed a large number of uncharacterized NRPS biosynthetic gene clusters (BGCs) and their products are underexplored. The majority of NRPSs remain silent potentially attributed to factors such as the low activity of the initiation unit or insufficient precursor supply. Exchanging the starter condensation (Cs) domains within initiation unit can change the length of acyl chains of NRPs, hinting at a promising strategy through swapping of a well-studied Cs domain to activate the initiation unit and harness primary metabolites as precursors, which may offer a new option to access silent BGCs. Here, we first pinpointed two highly efficient fusion sites for initiation unit exchanges. Subsequently, they were validated by replacing the initiation region of endopyrrole pathway with a Cs-containing initiation unit for generation of a lipo-endopyrrole derivate. We promptly leveraged this strategy of changing initiation unit to target six previously silent NRPS BGCs, three BGCs were successfully activated and five novel lipopeptides were identified, demonstrating its application to recover silent BGCs. Furthermore, we extended this strategy in more BGCs from different bacteria. Utilizing a heterologous Cs-containing unit to replace the initiation region of chitinimide biosynthetic pathway led to successful incorporation of N-terminal fatty acid chains into chitinimide to create artificial lipo-chitinimides. This study provides a feasible strategy to rationally recover silent BGCs and add fatty acid chains to NRPs, enriching the genome mining and combinatorial biosynthesis approach for bacterial natural products. SignificanceNonribosomal peptide synthetases (NRPSs) represent a valuable yet underexplored reservoir for bioactive natural products, but most of them are silent. This study introduces the concept that changing initiation unit to activate and optimize the functional expression of NRPSs, and successfully access three of six previously silent NRPS pathways, providing a feasible complement to the current genome mining approaches. Moreover, the N-terminal lipid chains are crucial for the activity of lipopeptides. We employed a heterologous Cs (starter condensation domain)-containing initiation unit to replace the original initiation region for creation of artificial nonribosomal lipopeptides, ultimately yielding three novel lipo-derivatives. This work presents a groundbreaking approach for activating and optimizing NRPSs and provides profound insight into the exploration of bacterial NRPS pathways.

synthetic biology↗

bioRxiv2025

A First-in-Class Therapy Targeting Transcription-Replication Conflicts Drives Synthetic Lethality in Homologous Recombination Deficient Cancers

Hidden beneath the layer of tumor malignancy lies unchecked proliferation driven by hyperactive transcription, which fosters the formation of RNA:DNA hybrids. These structural intermediates precipitate collisions between the transcription and replication machineries. Central to mitigating this genomic threat is interleukin enhancer-binding factor 2 (ILF2), which orchestrates the recruitment of RNA:DNA helicases to resolve R-loops and, in doing so, reveals cancer cells dependency on stress-mitigating mechanisms to avert genome catastrophe. Our discovery of Molephantin and its lead derivative, NYH0002 is the outcome of delineating ILF2s function. NYH002 exerts direct binding to the ILF2 complex and disrupts RNA:DNA helicase activity to elicit genome-wide DNA breakages. Tumors with elevated cyclin E and E2F1 expression are sensitive to NYH002, while those deficient in homologous recombination repair exhibit lethality. Collectively, these findings position ILF2 as a key regulator of R-loop homeostasis and reveal a therapeutic vulnerability that NYH002 exploits to suppress tumor malignancy.

cancer biology↗