Biology subjects

Gu, S.

Publications and source records attributed to Gu, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Scaling genetic discovery through automated post-GWAS interpretation

Genome-wide association studies (GWAS) have made association discovery systematic and cumulative, yet evidence for interpreting associated loci remains selective, heterogeneous and difficult to accumulate across studies. Here we present Post-GWAS Intelligence (PGI), which couples context-dependent post-GWAS analysis to a common representation of analysis-derived evidence. Its multi-agent engine, VariantAgent, executes workflows, records tool-derived results in standardized variant-centred units and generates trait-level interpretations linked to their analytical basis and evidential status. Complementary benchmarks showed that VariantAgent outperformed tested general-purpose and biomedical agents, with its largest advantage when success required both a correct answer and a valid analytical trace. Across 34 published GWAS, VariantAgent achieved a mean per-study recovery of 82.1% for directly comparable findings. The largest gains lay downstream of association mapping, where uniform analysis extended published interpretations and identified regulatory relationships at established risk loci not previously reported in the source studies. Analysis of 1,041 GWAS yielded 530,949 evidence units and corresponding reports, which PGI organized into a cross-trait catalogue. Catalogue interrogation resolved an apparent cross-trait overlap into distinct signals and trait-specific biological hypotheses. This work demonstrates how PGI can extend genetic discovery beyond association mapping by enabling biological evidence to accumulate across studies.

genetics↗

bioRxiv2026

Octave: Scale-resolved Evaluation of Spatial Gene Expression Prediction from Histology

Spatial gene expression prediction from histology is typically evaluated by the mean per-gene Pearson correlation (PCC). PCC does not distinguish spatial scales: coarse tissue organization alone can earn most of the score. A domain oracle makes this precise: given an image-derived partition of the tissue, the predictor that assigns each domain its measured mean expression is the best that any predictor constant on that partition can do. On a widely used benchmark with a ~100 um pitch, a learnable version that estimates each domain mean from training data reaches 82% of the PCC of a trained model, and a partition of the spot coordinates alone, given measured means, reaches 123%. At that pitch the score rewards coarse spatial structure whatever its source. We introduce OCTAVE, a scale-resolved evaluation that filters prediction and measurement into spatial bands with widths in micrometers and scores their agreement in each band. On 16 um Xenium data, differences that look modest under PCC grow at finer scales: at ~20 um the oracle shortfall relative to the trained model is 2.4 times its PCC shortfall at the median and exceeds it in all 13 specimens. Across 57 image encoders the finest band reorders 261 of the 1596 encoder pairs relative to PCC. The benchmark own finest band is 50 um wide, coarser than the scale at which these differences appear, so no score on that pitch can directly validate finer structure. OCTAVE reports how well a model predicts spatial expression at each spatial scale.

bioinformatics↗

bioRxiv2026

40S Ribosome Remodeling Triggers 18S rRNA U-tailing and DIS3L2-dependent Decay

Eliminating defective ribosomes through quality control is essential for accurate protein synthesis. However, the mechanisms that commit ribosomal subunits to decay remain poorly defined. Here, we identify a tandem mechanism in which ubiquitin-mediated ribosome remodeling and 18S rRNA uridylation lead to 40S ribosomal subunit decay. Specifically, we use an in vitro reconstitution system to show that the atypical kinase RIOK3 displaces the ribosomal protein eS26 from 40S subunits, exposing the 3 end of 18S rRNA. Nanopore direct RNA sequencing and motif analyses reveal that this remodeling event promotes oligo-uridylation, generating uridylated 18S rRNA decay intermediates. We further show that this uridylated 18S rRNA is selectively degraded by the 3-5 exoribonuclease DIS3L2. Moreover, DIS3L2-mediated exoribonucleolytic cleavage triggers endoribonucleolytic decay of the 18S rRNA, amplifying turnover. Together, our findings define a stepwise mechanism in which ribosome remodeling and RNA tailing commit defective 40S subunits to elimination, establishing a mechanistic framework for ribosome surveillance in mammalian cells.

molecular biology↗

bioRxiv2026

Targeting Macrophage Connexin 43 Hemichannels Suppresses Synovial Inflammation and Osteoarthritis

Osteoarthritis (OA) is the most common chronic joint disease and a leading cause of disability worldwide, yet no disease-modifying therapies are available. Although OA has traditionally been viewed as a cartilage-centered degenerative disease, growing evidence suggests that synovial inflammation is a critical driver of disease progression. However, the upstream mechanisms and actionable targets that govern this inflammatory response remain poorly defined. Connexin 43 (Cx43), a key regulator of musculoskeletal homeostasis that forms both gap junctions and hemichannels (HCs), is upregulated in osteoarthritic cartilage and synovium, but the specific contribution of Cx43 channels remains unclear. Here, we show that selective inhibition of Cx43 HCs attenuates OA progression in a murine post-traumatic OA model. Using adeno-associated virus (AAV)-mediated gene deletion, we found that Cx43 knockdown in joint tissues reduced cartilage erosion, synovial hyperplasia, subchondral bone sclerosis, and OA-associated pain. To dissect HC-specific functions, we developed a monoclonal antibody, Cx43(M1), that blocks Cx43 HCs without affecting junctional communication. Cx43(M1) preserved cartilage integrity, suppressed synovial inflammation, and alleviated pain behaviors, even when treatment was initiated after established cartilage damage. Mechanistically, the antibody preferentially localized to synovial macrophages, where it inhibited HC activity, reduced ATP and PGE2 release, and suppressed pro-inflammatory gene expression. Macrophage-specific Cx43 deletion recapitulated these protective effects, and Cx43 HC blockade in primary human OA synovial macrophages similarly inhibited inflammatory and catabolic responses. These findings identify synovial macrophage Cx43 HCs as upstream drivers of OA and support a paradigm shift from a predominantly chondrocyte-centered view toward synovial macrophage directed intervention.

immunology↗

bioRxiv2026

Elicitation of stem-directed antibodies in rhesus macaques by a conventional hemagglutinin immunogen

Because they can bind many strains of influenza, antibodies targeting the hemagglutinin (HA) stem have been attractive targets for vaccine development. Many monoclonal antibodies (mAbs) directed at the HA stem have been isolated from humans, and these mAbs have mediated broad protection in animal models. We describe here HA stem-directed mAbs isolated from rhesus macaques immunized with an "ordinary" H1 HA trimer. All immunized rhesus macaques developed high serum titers with broad reactivity to diverse H1N1 and H5N1 viruses, and 7 isolated mAbs strongly blocked canonical stem antibody CR6261 binding to H1. MAb DH726.1 robustly protected mice from lethal challenge with H1N1 and H5N1 viruses, and cryo-EM showed the binding footprint overlapped that of some human mAbs. These findings suggest that vaccination with the standard, trimeric HA immunogens may be sufficient to elicit stem antibodies at titers adequate to protect against zoonotic H5N1 influenza. In BriefEfforts to achieve broad influenza protection have largely emphasized increasingly sophisticated immunogen designs to redirect antibody responses toward conserved epitopes. Here, we show that a single, conventional HA immunogen readily elicits antibodies targeting the conserved HA stem, suggesting that routine influenza vaccination may provide broader protection than previously appreciated. HighlightsO_LIMonovalent HA immunization induces strong, stem-directed immune responses in rhesus macaques C_LIO_LIEpitope on the HA stem confirmed by Cryo-EM C_LIO_LIFc-mediated activation of immune effector cell by rhesus-derived stem mAb C_LIO_LIHA stem-directed antibody protects mice against H1N1 and H5N1 viruses C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/738984v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1535b59org.highwire.dtl.DTLVardef@192422dorg.highwire.dtl.DTLVardef@136b3d5org.highwire.dtl.DTLVardef@586fd4_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Near-Infrared II Scintillator for High-Resolution X-ray Imaging

Light scattering in scintillators is a pervasive problem and a key factor limiting X-ray imaging resolution. Here, we shift scintillator radioluminescence from the traditional visible range into the short-wave infrared (SWIR) or near-infrared II (NIR-II, 1000-3000 nm) window to mitigate light scattering and thereby enhance light penetration and X-ray imaging resolution. We present an NIR-II MgGa2O4:Ni2+ scintillator with peak emission at 1340 nm, achieving a threefold improvement in X-ray imaging resolution compared with visible scintillators owing to reduced light scattering. This heavy-metal-free NIR-II scintillator exhibits intense radioluminescence comparable to that of conventional visible-emitting CsI:Tl, achieving a detection limit of 56 nanograys per second, [~]100-fold lower than typical doses used in medical imaging. We show that this NIR-II scintillator enables high-resolution X-ray radiography of electronic circuit boards and biological tissues.

biophysics↗

bioRxiv2026

Targeted Photodegradation of Misfolded Proteins via Self-photosensitizing with Molecularly Produced Light

Misfolded proteins are tightly associated with various neurodegenerative diseases, and removing these misfolded proteins is one of the actively pursued approaches for seeking therapeutics for these diseases. In this study, we demonstrated that molecularly produced light (molecular light) from ADLumin-5, a self-photosensitizing chemiluminescence compound, could induce photo-oxidation and photodegradation of misfolded proteins, including beta-amyloid, tau, alpha-synucleins, and TDP-43 proteins in vitro. We validated the oxidation and degradation via LC-MS, MADLI-MS, and western blotting. Using beta-amyloid as a showcase, we demonstrated that, upon photo-oxidation and photodegradation, the toxicities of this misfolded protein were significantly reduced. To investigate the therapeutic effects of ADLumin-5 in vivo, we used the 5xFAD mouse model for longitudinal treatment for 4 months. In vivo molecular imaging results indicated that ADLumin-5 could reduce the accumulation of beta-amyloid proteins. Our study presents a novel approach to seek therapeutics for neurodegenerative disease via molecular light-induced degradation of misfolded proteins. In addition, because ADLumin-5 is dual-functional--enabling both photodegradation and in vivo imaging of misfolded protein changes--it can be considered a photo-theranostic agent for neurodegenerative diseases, representing a novel approach to drug discovery for neurodegenerative diseases.

neuroscience↗

bioRxiv2026

Distinct defense strategies against interbacterial antagonism underpin Acinetobacter persistence in polymicrobial environments

Despite intense interbacterial antagonism mediated by mechanisms such as the type VI secretion system (T6SS), pathogenic Acinetobacter frequently persist within highly competitive polymicrobial infections. How these pathogens navigate such hostile environments to achieve co-existence remains poorly understood. Here, we show that pathogenic Acinetobacter employ distinct, multifaceted strategies to resist elimination by both distantly related competitors and closely related kin. T6SS-active strains outcompete heterologous bacteria by deploying a large and diverse repertoire of antibacterial effectors. In contrast, some naturally T6SS-deficient strains resist exogenous T6SS attacks by elaborating a unique, high-density polysaccharide capsule (KL34) that functions as an effective physical shield. Most intriguingly, we uncover a mechanism that prevents lethal competition among kins. A conjugative multidrug-resistant plasmid encoding T6SS repressors is transferred into aggressive strains upon T6SS-mediated attack, leading to suppression of their antibacterial activity. We also found that conjugation is induced by T6SS attack, which effectively suppresses attacking kin and promotes co-infection in vivo. Together, these findings reveal a dynamic and multilayer defense program that enables Acinetobacter to withstand interbacterial warfare while facilitating co-existence. Our study establishes a paradigm in which damage-triggered plasmid transfer enforces targeted suppression, reshaping microbial interactions in polymicrobial communities.

microbiology↗

bioRxiv2026

Naturalistic behavior and self-generated neural activity predictive of self-correction.

Changing ones mind -- revising a past decision independently of external cues -- can lead to better outcomes, but its neural basis remains poorly understood. We therefore developed a challenging spatial task for rats where a conflict between innate foraging biases and task rules leads to abundant, spontaneous, and characteristically corrective changes-of-mind (COMs). Neural recordings in the hippocampus, a brain region implicated in counterfactual thinking, revealed two distinct stages wherein more local representations gave way to representations of distant alternatives after animals had begun to reverse course. These representations predicted their eventual choice, and often serially encoded trial start and end locations. Our novel task paradigm reveals distinct representational phases engaged during self-correction and uncovers a rich repertoire of hippocampal spatial representations tied to behavior.

neuroscience↗

bioRxiv2026

eBiota: Designing microbial communities from large seed pools with desired function using rapid optimization and deep learning

Designing microbial communities to generate target products is crucial for biotechnology, agriculture, and disease treatment. However, rationally designing such communities from large seed pools has become a major challenge, as the rapidly expanding number of complete microbial genomes greatly expands the search space and sharply increases the required screening time and computational cost. Here, we introduce eBiota, a platform for ab initio design of microbial communities from a pool of 21,514 strains to generate target products. eBiota not only identifies optimal strain combinations but also simulates community behaviors, including microbial interactions and relative abundances. eBiota integrates three modules: CoreBFS, a graph-based search algorithm that rapidly screens for bacteria with complete metabolic pathways related to the target product; ProdFBA, an extended flux balance analysis that identifies microbial consortia with maximal production efficiency; and DeepCooc, a deep learning model trained on 23,323 microbiome samples across various environments to infer co-occurrence patterns. We validated eBiotas capabilities in microbial community design and production efficiency calculation using public microbiome datasets, ranging from single strains to six-member consortia. Further in vitro experiments involving 94 strains confirmed eBiotas ability to identify species that inhibit pathogen growth and to accurately model the relative abundances within complex microbial communities. As an initial digital twin, eBiota provides a powerful platform for the rational design of functional microbial communities, offering new opportunities for metabolic engineering and synthetic biology.

bioengineering↗

bioRxiv2026

The world's first cloned golden wild yak via interspecific SCNT: 4800m donor origin and 4200m vitrified blastocyst transfer

Primarily inhabiting the harsh, high-altitude environment of the Qiangtang National Nature Reserve exceeding 5,000 meters above the sea (m.a.s.l.), the golden wild yak is critically endangered, with fewer than 300 individuals remaining in the world, a situation exacerbated by the significant challenges of conducting research and conservation of their genetic resources. Somatic cell nuclear transfer (SCNT) can be an effective method for their preservation, but facing several obstacles in this context, including the hypoxic stress at high altitude that impairs embryonic development due to in vitro manipulation, and constraints of long-distance embryo transport. In the present study, the ear tissue was collected from a childhood male golden wild yak at Xizang Geye Wildlife Rescue Station (4800 m.a.s.l.) and send to Institute of Animal Science at Beijing to derive fibroblast cells. Using fibroblast cells of the golden wild yak as nuclear donors, and bovine oocytes from a local slaughterhouse at Beijing as recipients, the interspecific SCNT (iSCNT) embryos were generated and in vitro developed to blastocysts. To maintain the embryonic viability after long-distance transportation from Beijing to Xizang, iSCNT blastocysts were subjected to cryopreservation by vitrification method. Thawing of vitrified iSCNT blastocysts were completed at Xizang Dangxiong Yak Breeding Innovation Base (4200 m.a.s.l.), and transferred into the uterine horn of domestic yaks. 257 days after blastocyst transfer, a cloned golden wild yak was successfully harvested on January 10, 2026. This work demonstrates, for the first time, that interspecies somatic cell nuclear transfer can successfully produce a cloned offspring under extreme conditions, spanning 4800 m.a.s.l. donor origin, long-distance vitrified embryo transportation, and high-altitude blastocyst transfer at 4200 m.a.s.l., establishing a viable strategy for conserving critically endangered high-altitude species.

genetics↗

bioRxiv2026

A Modular Framework for Automated Segmentation and Analysis of AFM Imaging of Chromatin Organization

Chromatin organization underlies essential genome functions, but its nanoscale organization remains challenging to capture and quantify with precision. Atomic force microscopy (AFM) offers direct structural readouts of DNA and chromatin, yet translating these rich images into reproducible biological metrics has been limited by the lack of standardized, scalable analysis tools. Here we present DNAsight, an automated analysis framework that integrates machine learning (ML)-based segmentation with modular, base-pair-calibrated quantification of DNA spatial organization, looping, nucleosome spacing, and protein clustering. Applied across diverse chromatin-associated proteins, DNAsight reveals protein-specific organizational signatures, including topology-dependent compaction by integration host factor (IHF), condition-dependent changes in loop-like DNA structures in cohesin-CTCF-precocious dissociation of sisters 5A (PDS5A) reactions, and promoter-driven multimerization of GAGA factor (GAF) clusters. The framework further enables direct extraction of nucleosome spacing distributions from raw AFM images, providing a label-free route to investigate chromatin fiber architecture. Together, these advances establish DNAsight as a generalizable and scalable approach for converting AFM measurements into quantitative insights into the physical principles of chromatin organization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/708946v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1225883org.highwire.dtl.DTLVardef@1e1041dorg.highwire.dtl.DTLVardef@1d53d18org.highwire.dtl.DTLVardef@9e0a2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Opposing regulatory logics converge on ABA receptors to govern the trade-off between growth and drought acclimation

Plants dynamically balance growth with stress responses through abscisic acid (ABA). How ABA signaling, but how ABA sensitivity is finely tuned is unclear. Here, we uncover a regulatory network of leucine-rich repeat receptor-like kinases (LRR-RLKs) that directly controls ABA receptors stability. We identify GASSHO1 (GSO1) as a core component that phosphorylates the PYL2 and PYL4 receptors, targeting them for degradation and thus serving as a brake on ABA signaling. Under drought stress, ABA accumulates and suppresses expression of GSO1-activating CIF peptides, releasing this brake to increase PYLs and sensitivity. This mechanism opposes our previously identified CEPR2 pathway, where drought-induced CEP peptides inhibit the kinase to stabilize PYLs. The integration of these two antagonistic modules within an LRR-RLK network enables precise, dynamic control of ABA perception. This work reveals a molecular framework explaining how plants calibrate the critical balance between growth and drought acclimation.

molecular biology↗

bioRxiv2026

Low levels of metabolic auxotrophy among environmental Pseudomonas isolates

Auxotrophy, the inability of bacteria to synthesize one or multiple essential metabolites (e.g. amino acids, vitamins, metabolites) is thought to be common among bacteria. However, studies often rely either on bioinformatic tools to predict auxotrophies from genome data or on experiments with low numbers of strains. Here, we combine experimental and bioinformatic approaches to assess amino acid auxotrophy levels among 315 co-isolated natural Pseudomonas strains from pond and soil habitats. Both approaches revealed that Pseudomonas isolates are predominantly prototrophs. We identified one single histidine auxotroph and five non-specific auxotrophs featuring complex growth phenotypes incompatible with single amino acid auxotrophies. While different bioinformatic pipelines vary in the extent to which auxotrophy is over- or underestimated, none of the pipelines could resolve the basis of non-specific auxotrophies. Our analysis further revealed the existence of multiple alternative biosynthesis pathways for methionine, proline, and phenylalanine, with significant enrichments of specific pathways among soil or pond strains. We conclude that combining experiments with bioinformatics is a powerful approach to assess the metabolic potential of environmental bacteria. Moreover, taxa like Pseudomonas can be predominantly prototrophic possibly owing to their generalist lifestyle, thus calling for nuanced ecological concepts predicting auxotrophy levels based on lifestyle and habitat.

microbiology↗

bioRxiv2026

A multi-agent platform for assessment and improvement of bioinformatics software documentation

Rapid advances in bioinformatics have transformed biomedical research in areas such as single-cell and spatial omics, digital pathology, and multi-modal data integration, yet software usability and reproducibility have not kept pace with the growing complexity and proliferation of computational tools. Inconsistent, incomplete, or inaccessible documentation remains a pervasive and underappreciated barrier, limiting tool adoption, hindering reproducibility across laboratories, and reducing the long-term impact of computational methods. Here, we introduce BioGuider, a multi-agent platform designed to systematically evaluate and improve documentation quality in bioinformatics software. Rather than treating documentation as ancillary text, BioGuider models it as a first-class, testable object. The platform implements a modular pipeline for documentation collection, assessment, reporting, and optional correction, with specialized agents that emulate real-world user interactions. BioGuider evaluates documentation against standardized, task-oriented criteria spanning installation, configuration, usage, and tutorials, and supports iterative, constraint-aware refinement while preserving code integrity and biological context. We benchmark BioGuider using a controlled error-injection framework that introduces realistic documentation failures across general, biology-specific, and configuration-related categories. Across multiple large language models, BioGuider demonstrates robust error detection and correction, with strong performance maintained under severe documentation degradation. Applying BioGuider to 47 widely used bioinformatics tools, we observe a positive association between documentation quality and citation frequency, highlighting documentation as a previously under-quantified driver of software adoption and scientific impact.

bioinformatics↗

bioRxiv2026

Fine-tuned synthetic transcription factors for production of 3' phosphoadenosine-5'-phosphosulfate in yeast

Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for producing various natural products. Balancing multi-enzyme expression, while maintaining robust microbial growth, remains a limiting factor for engineering long biosynthetic pathways in yeast. Here, we improved the transcriptional capacity of our previously developed isopropyl {beta}-D-1-thiogalactopyranoside (IPTG)-inducible synthetic transcription factors (synTFs) derived from the plant JUB1 DNA-binding domain. To this end, at cysteine positions within surface-exposed loop regions of a JUB1-derived DNA-binding scaffold, we introduced a short peptide to enhance loop flexibility while providing local stability and orientation. The generated synTFs, so-called JUB1-X synTFs, varying in strength, have been successfully used to improve the synthesis of 3-phosphoadenosine 5-phosphosulfate (PAPS), a universal sulfate donor necessary for the synthesis of bioactive molecules, including therapeutic glycosaminoglycans and sulfolipids. Using only this engineered yeast strain in simple batch culture, PAPS accumulation of 21.4 {+/-} 5.8 mg g-1 cdw was achieved after only 5 hours of inducing the expression of JUB1-X synTFs. Beyond PAPS production, the design principle demonstrated here provides a generalizable strategy to fine-tune other plant-derived synTFs, expanding the regulatory capabilities of existing synTF collections. Together, this work offers a modular, scalable approach to constructing high-performance gene circuits and supports the development of yeast cell factories for complex metabolic and synthetic biology applications.

synthetic biology↗

bioRxiv2025

Discovery of Abundant Nano-scale Lymphatic-like Vessels in Brains

As one of the most metabolically active organs, the brain requires an exceptionally efficient system for waste clearance to sustain its high metabolic demands. However, whether such a system exists--and, if so, what structural features enable its efficiency--remains incompletely understood. More than a decade ago, the "glymphatic system" was proposed to describe neurofluid transport through cerebrospinal fluid (CSF) and perivascular spaces (PVS), in conjunction with dural and meningeal lymphatic pathways. Nevertheless, it remains unresolved whether neurofluid transport is organized as a structured, vessel-like flow network. Moreover, in stark contrast to the dense network of blood capillaries in the brain, only a sparse population of lymphatic vessels has been identified, raising doubts as to whether the currently recognized lymphatic architecture alone can support efficient metabolic waste clearance. By combining expansion microscopy with CRANAD-3, a pan-{beta}-amyloid fluorescent probe, we discovered abundant nanoscale lymphatic-like vessels (NLVs) within the brain parenchyma of both mice and humans. The majority of these structures have diameters below 1,000 nm and exhibit moderate positivity for multiple lymphatic markers, including LYVE-1, Prox-1, PDPN, and VEGFR3. NLVs frequently coil around blood vessels, and putative connections between vascular structures were observed. Notably, some NLVs traverse multiple cortical layers and display distinct orientation patterns that vary across cortical laminae. This discovery reveals a previously "hidden" vascular network in the brain parenchyma and raises the possibility that an abundant, highly organized system of nanoscale tubular structures may provide an efficient conduit for metabolic waste clearance. Such a system could represent a critical, previously unrecognized component supporting the brains extraordinary metabolic demands.

neuroscience↗

bioRxiv2025

Demonstrating and engineering the de novo synthesis of two aromatic chemicals estragole and anethole in Escherichia coli

Estragole and anethole are the major flavoring and aroma components of the essential oils extracted from basil (Ocimum basilicum L.), anise (Pimpinella anisum), star anise (Illicium verum), and fennel (Foeniculum vulgare). Besides, they also exhibit bioactivities, such as antioxidation, antimicrobial, immunomodulation, inti-inflammation, and anti-diabetes. These properties make them being faced with substantial applications in the industries of food, cosmetics, pharmaceutics, and agriculture. Commercial estragole and anethole are currently obtained from extraction from their original plants, which suffers from unstable quality and resources. Microbial synthesis is a promising alternative approach to sustainably obtain estragole and anethole, but has not been achieved. In this study, we modularly redesigned the synthetic pathways based on the Escherichia coli chassis. Firstly, a p-coumaryl alcohol over-accumulator (436.28 mg/L) was engineered to facilitate the validation of the undemonstrated downstream module. De novo biosynthesis of estragole and anethole was achieved by stepwise analysis and BioBricks assembly. The intracellular availability of S-adenosyl-L-methionine (SAM) was revealed to be the key limiting factor, and was enhanced by deregulating the synthesis. Finally, the productions of estragole and anethole were improved by 246.88% and 202.44% compared with the initial strains. This study represents the first synthesis of estragole and anethole in microbial chassis, and the demonstration of microbial synthesis of propenyl phenols. Besides, it provided an alternative sustainable approach to obtain flavoring and aroma chemicals.

synthetic biology↗