Biology subjects

Zhang, W.

Publications and source records attributed to Zhang, W..

At least 559 records · Page 31Linked to original sources

bioRxiv2023

Contrasting roles of different mismatch repair proteins in basal-like breast cancer

The mismatch repair (MMR) pathway is known as a tumor suppressive pathway and genes involved in MMR are commonly mutated in hereditary colorectal or other cancer types. However, the function of MMR genes/proteins in breast cancer progression and metastasis are largely undefined. We found that MSH2, but not MLH1, is highly enriched in basal-like breast cancer (BLBC) and that its protein expression is inversely correlated with overall survival time (OS). MSH2 expression is frequently elevated due to genomic amplification or gain-of-expression in BLBC, which results in increased MSH2 protein to pair with MSH6 (collectively referred to as MutS). Genetic deletion of MSH2 or MLH1 results in a contrasting phenotype in metastasis, with MSH2-deletion leading to reduced metastasis and MLH1-deletion to enhanced liver or lung metastasis. Mechanistically, MSH2 - but not MLH1 - binds to the promoter region of interferon receptor 1 (IFNAR1) and suppresses its expression in BLBC. Deletion of MSH2 initiates a chain of immune reactions via the upregulation of IFNAR1 expression and the activation of type 1 interferon signaling, which explains a highly immune active tumor microenvironment in tumors with MSH2-deficiency. Our study supports the contrasting functions of MSH2 and MLH1 in BLBC progression and metastasis due to the differential regulation of IFNAR1 expression, which challenges the paradigm of the MMR pathway as a universal tumor suppressive mechanism.

cancer biology↗

bioRxiv2023

Insight into the structures of unusual base pairs in RNA complexes containing primer/template/adenosine ligand

In prebiotic RNA world, the self-replication of RNA without enzymes can be achieved through the utilization of 2-aminoimidazole activated nucleotides as efficient substrates. The mechanism of RNA nonenzymatic polymerization has been extensively investigated biophysically and structurally by using the model of RNA primer/template complex which is bound by the imidazolium-bridged or triphosphate-bridged diguanosine intermediate. However, beyond the realm of guanosine substrate, the structural insight into how alternative activated nucleotides bind and interact with RNA primer/template complex remains unexplored, which is important for understanding the low reactivity of adenosine and uridine substrates in RNA primer extension, as well as its relationship with the structures. Here we use crystallography as method and determine a series of high-resolution structures of RNA primer/template complexes bound by ApppG, the close analog of dinucleotide intermediate containing adenosine and guanosine. The structures show that ApppG ligands bind to RNA template through both Watson-Crick and noncanonical base pairs, with the primer 3'-OH group far from the adjacent phosphorus atom of the incoming substrate. The structures indicate that, when adenosine is included in the imidazolium-bridged intermediate, the complexes are likely preorganized in a suboptimal conformation, making it difficult for the primer to in-line attack the substrate. Moreover, by cocrystallizing the RNA primer/template with chemically activated adenosine and guanosine monomers, we successfully observe the slow formation of the imidazolium-bridged intermediate (Ap-AI-pG) and the preorganized structure for RNA primer extension. Overall, our studies offer a structural explanation for the slow rate of RNA primer extension when using adenosine-5-phosphoro-2-aminoimidazolide as a substrate during nonenzymatic polymerization.

biochemistry↗

bioRxiv2023

No evidence for the association between COVID-19 and neuroinflammation: A diffusion basis spectrum imaging study.

COVID-19 remains a significant international public health concern. Yet, the mechanisms through which symptomatology emerges remain poorly understood. While SARS-CoV-2 infection may induce prolonged inflammation within the central nervous system, the evidence primarily stems from limited small-scale case investigations. To address this gap, our study capitalized on longitudinal UK Biobank neuroimaging data acquired prior to and following COVID-19 testing (N=416 including n=224 COVID-19 cases; Mage=58.6). Putative neuroinflammation was assessed in gray matter structures and white matter tracts using non-invasive Diffusion Basis Spectrum Imaging (DBSI), which estimates inflammation-related cellularity (DBSI-restricted fraction; DBSI-RF) and vasogenic edema (DBSI-hindered fraction; DBSI-HF).We hypothesized that COVID-19 case status would be associated with increases in DBSI markers after accounting for potential confound (age, sex, race, body mass index, smoking frequency, and data acquisition interval) and multiple testing. COVID-19 case status was not significantly associated with DBSI-RF (|{beta}|s<0.28, pFDR >0.05), but with greater DBSI-HF in left pre- and post-central gyri and right middle frontal gyrus ({beta}s>0.3, all pFDR=0.03). Intriguingly, the brain areas exhibiting increased putative vasogenic edema had previously been linked to COVID-19-related functional and structural alterations, whereas brain regions displaying subtle differences in cellularity between COVID-19 cases and controls included regions within or functionally connected to the olfactory network, which has been implicated in COVID-19 psychopathology. Nevertheless, our study might not have captured acute and transitory neuroinflammatory effects linked to SARS-CoV-2 infection, possibly due to symptom resolution before the imaging scan. Future research is warranted to explore the potential time- and symptom-dependent neuroinflammatory relationship with COVID-19.

neuroscience↗

bioRxiv2023

Theranostic Bottle-Brush Polymers Tailored for Universal Solid-Tumour Targeting

Nanomedicines involving nanotechnologies and engineering of nanomaterials for medicines have shown great promise in diagnosis and treatment of diseases including cancers. A major hurdle that limits the successful clinical translation of nanomedicines, however, is how to overcome the cascaded biological barriers and improve the delivery efficacy towards the disease sites and minimize the toxicity against healthy tissues and cells. Here, we report a type of bottle-brush-like polymers systematically optimized in their chemical structures, sizes, and surface charges that lead to their outstanding pharmacokinetics and tumour-targeting performances in a variety of both subcutaneous and orthotopic tumour models. The potential mechanism has been studied by revealing the structure-activity relationship of these polymers in overcoming the biological barriers, including their avoidance by the immune system and deep tumour infiltration. Our study may offer insight for a rational design of highly efficient delivery platform of polymeric nanomedicines that could effectively overcome the cascaded biological barriers and thus lead to high tumour-targeting efficacy and low toxicity.

bioengineering↗

bioRxiv2023

Ladder-like multimerization of FoxP3 enables microsatellite recognition and DNA bridging

FoxP3 is a transcription factor (TF) essential for development of regulatory T cells (Tregs), a branch of T cells that suppress excessive inflammation and autoimmunity1-5. Molecular mechanisms of FoxP3, however, remain elusive. We here show that FoxP3 utilizes the Forkhead domain--a DNA binding domain (DBD) that is commonly thought to function as a monomer or dimer--to form a higher-order multimer upon binding to TnG repeat microsatellites. A cryo-electron microscopy structure of FoxP3 in complex with T3G repeats reveals a ladder-like architecture, where two double-stranded DNA molecules form the two "side rails" bridged by five pairs of FoxP3 molecules, with each pair forming a "rung". Each FoxP3 subunit occupies TGTTTGT within the repeats in the manner indistinguishable from that of FoxP3 bound to the Forkhead consensus motif (FKHM; TGTTTAC). Mutations in the "intra-rung" interface impair TnG repeat recognition, DNA bridging and cellular functions of FoxP3, all without affecting FKHM binding. FoxP3 can tolerate variable "inter-rung" spacings, explaining its broad specificity for TnG repeat-like sequences in vivo and in vitro. Both FoxP3 orthologs and paralogs show similar TnG repeat recognition and DNA bridging. These findings thus reveal a new mode of DNA recognition that involves TF homo-multimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.

biochemistry↗

bioRxiv2023

Pan-genome and Haplotype Map of Cultivars and Their Wild Ancestors Provides Insights into Adaptive Evolution of Cassava (Manihot esculenta Crantz)

Cassava is the most important starch sources, a tropical model crop. We constructed nearly T2T genomes of cultivar AM560, wild ancestors FLA4047 and W14, pan-genome of 24 representatives and a clarified evolutionary tree with 486 accessions. Comparison of SVs and SNVs between the ancestors and cultivated cassavas revealed predominant expansion, contraction of genes and gene families. Significantly selective sweeping occurred in the cassava genomes in 122 footprints with 1,519 candidate domestication genes. We identified selective mutations in MeCSK and MeFNR3 promoting photoreaction associated with MeNADP-ME of C4 assimilation in modern cassava. Co-evolved retardation of floral primordia and initiation of storage roots arose from MeCOL5 mutants with altered bindings to MeFT1, MeFT2 and MeTFL2. MebHLHs evolved to regulate the biosynthesis, transport and endogenous remobilization of cyanogenic glucosides, with new functionalities of MeMATE1, MeGTR in selected sweet cassava. These findings enhanced comprehensive knowledge and database on the evolution and breeding of cassava. HIGHLIGHTSO_LIThree nearly T2T cassava genomes of cultivar AM560 and its wild ancestors FLA4047 and W14. C_LIO_LIA species-level cassava panSV haplotype map across 346,322 structural variations over 31,362 gene families and 96,032,008 SNPs and InDels variations globally and a clarified evolutionary tree with 486 accessions. C_LIO_LISelective mutations in MeCSK and MeFNR3 promoted photoreaction associated with MeNADP-ME of C4 assimilation shaped the C3-C4 intermediate photosynthesis of modern cassava. C_LIO_LICoevolution of floral primordia contrary to initiated storage root is pivotal for the domestication of cassava, and arose from MeCOL5 mutants altered the binding with MeFT1, MeFT2 (SP6A), and MeTFL2. C_LI

systems biology↗

bioRxiv2023

Polygenic basis of strong and rapid flowering time response to environment perturbations in wild Arabidopsis thaliana population

Despite the importance in understanding the impact of climate change, the genetics of rapid response to changing environments and its role in adaptive evolution remains elusive. Here, we studied flowering time response to environment changes using 514 Arabidopsis thaliana worldwide accessions with re-sequencing genomes and flowering time measurements from ten unique environments with variable temperature, drought, daylight and competition stresses. We revealed a polygenic basis of flowering time mean and plasticity, underpinned by 52 mean and plasticity QTL. Widespread interaction between mean QTL, polygenic background and surrounding environments considerably altered the amount of additive genetic variance and allelic effects of detected QTL. This caused variability in phenotype plasticity and across environment variation in genetic variance, resulting in rapid flowering time response to environment perturbations. In addition, the plastic alleles showed a higher correlation with the environment factors than that from randomly sampled alleles, suggesting a potential role in climate adaptation. We therefore proposed a polygenic interaction model, whereby large effect QTL and polygenic background simultaneously interacted with the surrounding environment, underlying rapid response to changing environments. Results from our study thus provided deeper insights into the genetics of plasticity, with potential benefit in genomic selection of crops in heterogeneous environments and predicting changes in species distribution and the evolutionary trajectory of wild populations.

genetics↗

bioRxiv2023

Genomic context sensitizes regulatory elements to genetic disruption

Enhancer function is frequently investigated piecemeal using truncated reporter assays or single deletion analysis. Thus it remains unclear to what extent enhancer function at native loci relies on surrounding genomic context. Using the Big-IN technology for targeted integration of large DNAs, we analyzed the regulatory architecture of the murine Igf2/H19 locus, a paradigmatic model of enhancer selectivity. We assembled payloads containing a 157-kb functional Igf2/H19 locus and engineered mutations to genetically direct CTCF occupancy at the imprinting control region (ICR) that switches the target gene of the H19 enhancer cluster. Contrasting activity of payloads delivered at the endogenous Igf2/H19 locus or ectopically at Hprt revealed that the Igf2/H19 locus includes additional, previously unknown long-range regulatory elements. Exchanging components of the Igf2/H19 locus with the well-studied Sox2 locus showed that the H19 enhancer cluster functioned poorly out of context, and required its native surroundings to activate Sox2 expression. Conversely, the Sox2 locus control region (LCR) could activate both Igf2 and H19 outside its native context, but its activity was only partially modulated by CTCF occupancy at the ICR. Analysis of regulatory DNA actuation across different cell types revealed that, while the H19 enhancers are tightly coordinated within their native locus, the Sox2 LCR acts more independently. We show that these enhancer clusters typify broader classes of loci genome-wide. Our results show that unexpected dependencies may influence even the most studied functional elements, and our synthetic regulatory genomics approach permits large-scale manipulation of complete loci to investigate the relationship between locus architecture and function. HIGHLIGHTSO_LIComposite enhancer elements are subject to genomic context effects mapped to a specific architecture of their endogenous loci. C_LIO_LIIgf2/H19 expression is affected by long-range regulatory elements beyond the canonically defined locus, and the H19 enhancer cluster in particular relies on the surrounding context at its endogenous locus. C_LIO_LIThe Sox2 LCR functions as an autonomous enhancer without requiring additional surrounding context. C_LIO_LIThe influence of genomic context is buffered at intact loci, but manifests more strongly as key regulatory elements are deleted or repositioned. C_LI

genomics↗

bioRxiv2023

Exploiting the therapeutic vulnerability of IDH-mutant gliomas with zotiraciclib

Isocitrate dehydrogenase (IDH)-mutant gliomas have distinctive metabolic and biological traits that may render them susceptible to targeted treatments. Here, by conducting a high-throughput drug screen, we pinpointed a specific susceptibility of IDH-mutant gliomas to zotiraciclib (ZTR). ZTR exhibited selective growth inhibition across multiple IDH-mutant glioma in vitro and in vivo models. Mechanistically, ZTR at low doses suppressed CDK9 and RNA Pol II phosphorylation in IDH-mutant cells, disrupting mitochondrial function and NAD+ production, causing oxidative stress. Integrated biochemical profiling of ZTR kinase targets and transcriptomics unveiled that ZTR-induced bioenergetic failure was linked to the suppression of PIM kinase activity. We posit that the combination of mitochondrial dysfunction and an inability to adapt to oxidative stress resulted in significant cell death upon ZTR treatment, ultimately increasing the therapeutic vulnerability of IDH-mutant gliomas. These findings prompted a clinical trial evaluating ZTR in IDH-mutant gliomas towards precision medicine (NCT05588141). HighlightsO_LIZotiraciclib (ZTR), a CDK9 inhibitor, hinders IDH-mutant glioma growth in vitro and in vivo. C_LIO_LIZTR halts cell cycle, disrupts respiration, and induces oxidative stress in IDH-mutant cells. C_LIO_LIZTR unexpectedly inhibits PIM kinases, impacting mitochondria and causing bioenergetic failure. C_LIO_LIThese findings led to the clinical trial NCT 05588141, evaluating ZTR for IDH-mutant gliomas. C_LI

cancer biology↗

bioRxiv2023

SPPUSM: An MS/MS spectra merging strategy for improved low-input and single-cell proteome identification

Single and rare cell analysis provides unique insights into the investigation of biological processes and disease progress by resolving the cellular heterogeneity that is masked by bulk measurements. Although many efforts have been made, the techniques used to measure the proteome in trace amounts of samples or in single cells still lag behind those for DNA and RNA due to the inherent non-amplifiable nature of proteins and the sensitivity limitation of current mass spectrometry. Here, we report an MS/MS spectra merging strategy termed SPPUSM (same precursor-produced unidentified spectra merging) for improved low-input and single-cell proteome data analysis. In this method, all the unidentified MS/MS spectra from multiple test files are first extracted. Then, the corresponding MS/MS spectra produced by the same precursor ion from different files are matched according to their precursor mass and retention time (RT) and are merged into one new spectrum. The newly merged spectra with more fragment ions are next searched against the database to increase the MS/MS spectra identification and proteome coverage. Further improvement can be achieved by increasing the number of test files and spectra to be merged. Up to 18.2% improvement in protein identification was achieved for 1 ng HeLa peptides by SPPUSM. Reliability evaluation by the "entrapment database" strategy using merged spectra from human and E. coli revealed a marginal error rate for the proposed method. For application in single cell proteome (SCP) study, identification enhancement of 28%-61% was achieved for proteins for different SCP data. Furthermore, a lower abundance was found for the SPPUSM-identified peptides, indicating its potential for more sensitive low sample input and SCP studies.

molecular biology↗

bioRxiv2023

Engineering transposon-associated TnpB-1 ωRNA system for efficient gene editing and disease treatment in mouse

Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (409 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA ({omega}RNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-{omega}RNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and prevented the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues.

molecular biology↗

bioRxiv2023

A statistical method for image-mediated association studies discovers genes and pathways associated with four brain disorders

Brain imaging and genomics are critical tools enabling characterization of the genetic basis of brain disorders. However, imaging large cohorts is expensive, and may be unavailable for legacy datasets used for genome-wide association studies (GWAS). Using an integrated feature selection/aggregation model, we developed Image-Mediated Association Study (IMAS), which utilizes borrowed imaging/genomics data to conduct association mapping in legacy GWAS cohorts. By leveraging the UK Biobank image-derived phenotypes (IDPs), IMAS discovered genetic bases underlying four neuropsychiatric disorders and verified them by analyzing annotations, pathways, and expression Quantitative Trait Loci (eQTLs). A cerebellar-mediated mechanism was identified to be common to the four disorders. Simulations show that, if the goal is identifying genetic risk, IMAS is more powerful than a hypothetical protocol in which the imaging results were available in the GWAS dataset. This implies the feasibility of reanalyzing legacy GWAS datasets without conducting additional imaging, yielding cost-savings for integrated analysis of genetics and imaging.

bioinformatics↗

bioRxiv2023

Electrically Conductive DNA-Inspired Coating for Intracortical Neural Microelectrodes

Different from conventional electrodes, intracortical neural microelectrodes are the size of one or several neural cells. Due to the limited space for cell-electrode connections, the bio-integration between each neural cell and the electrode surface is critical. To improve bio-integration and electrode functions, various coating materials (such as conductive polymers (CPs), carbon nanotubes (CNTs), and natural hydrogels) have been developed aiming to provide an enhanced interface for neuron recruitment, bio-anchorage, and electrical function. However, synthetic materials usually have limited biocompatibility and/or relatively high cytotoxicity, while biological materials present poor electrical functions. Therefore, current coatings possess biological, functional or electrochemical limitations that are not optimal for intracortical neural microelectrodes. To overcome this obstacle, we developed an electrically conductive coating based on biological molecules, named Janus base nano-coating (JBNc). JBNc is formed by Janus base nanotubes (JBNts) which are a family of nanotubes assembled from engineered DNA base pair units. Based on the long-distance translocation ability of the {pi} electrons of JBNts, we developed them into an electrically conductive coating on the electrode surface. For the first time, we reported the DNA-inspired JBNc had an electrochemical performance that met and exceeded standard metal electrode surface in cyclic voltammetry, impedance spectroscopy, charge injection capacity tests, and neural recording. Moreover, we demonstrated enhanced bio-anchorage and microelectrode interface integration using SEM and AFM. Importantly, we demonstrated enhanced functional response to JBNc microelectrodes with immunohistochemical staining and RNA sequencing (RNAseq) analysis. Using cell viability assays, we also showed the benefits of DNA-mimicking chemistry of Janus base nanomaterials compared to conventional microelectrode coatings. We anticipate that these results will serve as a foundation for the continued development and study of JBNc to enhance interface dynamics and ultimately the performance and reliability of brain microelectrodes.

bioengineering↗

bioRxiv2023

snoRNA-guided tRNA 2'-O-methylation controls codon-biased gene expression and cellular states

snoRNAs are a large family of noncoding (nc)RNAs present across eukaryotes and archaea. While a subset of them guide 2-O- methylation (Nm) and pseudouridylation ({Psi}) of rRNAs and snRNAs, targets of most snoRNAs remain unknown. Here we used PARIS2 to map snoRNA targets, revealing an extensive and conserved snoRNA-tRNA interaction network. Using optimized denatured RiboMeth-seq (dRMS), we discovered snoRNA-guided Nm sites in ncRNAs, including tRNAs. Loss of snoRNAs and their associated 2-O-methyltransferase FBL reduced tRNA modifications and increased fragmentation. CRISPR knockout of the D97/D133 family of snoRNAs reduced the activity and levels of several target tRNAs, including elongator (e)Met-CAU, leading to codon-biased transcriptome and translatome in human cells. The codon-biased gene expression tipped the balance between the dichotomous cellular states of proliferation and differentiation, and skewed germ layer potential of mouse embryonic stem cells. Together, we discovered a snoRNA-guided tRNA modification mechanism controlling codon-biased gene expression and cellular states.

biochemistry↗

bioRxiv2023

EquiScore: A generic protein-ligand interaction scoring method integrating physical prior knowledge with data augmentation modeling

Developing robust methods for evaluating protein-ligand interactions has been a long-standing problem. Here, we propose a novel approach called EquiScore, which utilizes an equivariant heterogeneous graph neural network to integrate physical prior knowledge and characterize protein-ligand interactions in equivariant geometric space. To improve generalization performance, we constructed a dataset called PDBscreen and designed multiple data augmentation strategies suitable for training scoring methods. We also analyzed potential risks of data leakage in commonly used data-driven modeling processes and proposed a more stringent redundancy removal scheme to alleviate this problem. On two large external test sets, EquiScore outperformed 21 methods across a range of screening performance metrics, and this performance was insensitive to binding pose generation methods. EquiScore also showed good performance on the activity ranking task of a series of structural analogs, indicating its potential to guide lead compound optimization. Finally, we investigated different levels of interpretability of EquiScore, which may provide more insights into structure-based drug design.

bioinformatics↗

bioRxiv2023

Enzyme-mediated alkynylation enables transcriptome-wide identification of pseudouridine modifications

Pseudouridine ({Psi}) is one of the most abundant chemical modifications that exists in various types of RNA species and is known to play important roles in RNA function. The advances in studies of {Psi} in less abundant messenger RNA species have been hindered by a lack of suitable methods to precisely and sensitively map their distributions. Here we show that a methyltransferase from Methanocaldococcus jannaschii can label RNA {Psi} efficiently and specifically with various functional groups, both in isolated RNA and inside cells. We leveraged this enzymatic labeling strategy to develop ELAP-seq as a facile method to enrich {Psi}-modified transcripts for the detection of {Psi} modifications at single base resolution with high sensitivity and low background. Using this method, we identified over 10, 000 candidate {Psi} sites from human transcripts, which provides new insights into {Psi} biosynthesis and function. Our study provides a chemical biology method that specifically labels {Psi} for its detection and functional alteration.

biochemistry↗

bioRxiv2023

Processing and Storage Methods Affect Oral and Gut Microbiome Composition

Across microbiome studies, fecal and oral samples are stored and processed in different ways, which could affect the observed microbiome composition. Here, we compared treatment methods, which included both storage conditions and processing methods, applied to samples prior to DNA extraction to determine how each affects microbial community diversity as assessed by 16S rRNA gene sequencing. We collected dental swab, saliva, and fecal samples from 10 individuals, with three technical replicates per treatment method. We assessed four methods of processing fecal samples prior to DNA extraction. We also compared different fractions of frozen saliva and dental samples to fresh samples. We found that lyophilized fecal samples, fresh whole saliva samples, and the supernatant fraction of thawed dental samples retained the highest levels of alpha diversity in samples. The supernatant fraction of thawed saliva samples had the second highest alpha diversity compared to fresh. Then we investigated the differences in microbes between different treatments at the domain and phylum levels as well as identified the amplicon sequence variants (ASVs) that were significantly different between the methods producing the highest alpha diversity and the other treatment methods. Lyophilized fecal samples had a greater prevalence of Archaea as well as a greater ratio of Firmicutes to Bacteroidetes compared to the other treatment methods. Our results provide practical considerations, not only for selection of processing method, but also for comparing results across studies that use these methods. Our findings also indicate differences in treatment method could be a confounding factor influencing the presence, absence, or differential abundance of microbes reported in conflicting studies.

microbiology↗

bioRxiv2023

Haplotype-phased genome revealed the butylphthalide biosynthesis and hybrid origin of Ligusticum chuanxiong

Butylphthalide, one type of phthalides, is one of the first-line drugs for ischemic stroke therapy, while no enzyme involved in its biosynthesis pathway has been reported. Here, we present the first haplotype-resolved genome of Ligusticum chuanxiong Hort., a long-cultivated and phthalide-rich medicinal plant in Apiaceae. Based on comprehensive candidate gene screening, four Fe (II)- and 2-oxoglutarate-dependent dioxygenases (2OGDs) and two CYPs were mined and further biochemically verified as phthalide C-4/C-5 desaturase (P4,5Ds) that converts senkyunolide A to l-n-butylphthalide (l-NBP) and ligustilide to butylidenephthalide. The substrate promiscuity and functional redundancy featured for P4,5Ds may contribute to the high phthalide diversity in L. chuanxiong. Notably, comparative genomic evidence supported L. chuanxiong as a diploid hybrid with L. sinense as a potential parent. The two haplotypes demonstrated exceptional structure variance and diverged around 3.42 million years ago (Ma). Our study is an icebreaker for the dissection of phthalide biosynthesis pathway and reveals the hybrid origin of L. chuanxiong. These findings will facilitate the future metabolic engineering for l-NBP production and breeding efforts for L. chuanxiong.

synthetic biology↗