Science China Life Sciences

Virus-like particles (VLPs) enable transient, non-integrating delivery of CRISPR-Cas9 ribonucleoprotein cargo. Although VLPs have been reported for efficient DNA editing via base editors RNP delivery, the diversity of base editors tested as VLPs remains limited. We generated and benchmarked a panel of 12 base editors on the v5 eVLP backbone, targeting three genomic loci (HEK3, B2M, PDCD1) across five VLP dosages in LentiX-293T cells. Editing efficiency was generally dosage-dependent across all editors and varied by editor class and identity; PAM-flexible variants had lower editing efficiency than NGG-restricted counterparts, and the dual-function SPACE base editors showed reduced efficiency. We further characterized position-specific editing efficiencies and outcomes of the base editor VLP collection, revealing that a wide variety of mutation types are possible with the base editors in this collection.

Tomato yellow leaf curl virus (TYLCV) is a devastating viral pathogen threatening agricultural crops globally. In this study, we identified a novel TYLCV isolate (TYLCV-YN6244), which caused viral epidemic in resistant tomato cultivars at Yuanmo county, Yunnan Province of China. We determined the complete genome of TYLCV-YN6244 and found it encoded six viral proteins characteristic of Geminivirus. We identified its V2 protein as a potent viral suppressor of RNA silencing (VSR), and generated infectious clone of wildtype TYLCV-YN6244, or V2-defective TYLCV-YN6244 (TYLCV-YN6244-{Delta}V2) in which V2 was deleted. Both of infectious clones were capable of systemically infecting tobacco and tomato. However, TYLCV-YN6244 but not TYLCV-YN6244-{Delta}V2 could cause disease symptoms in wildtype tobacco or tomato plants, and viral accumulation was drastically reduced in plants infected with TYLCV-YN6244-{Delta}V2 compared to TYLCV-YN6244 while the efficiency of virus-derived small interfering RNAs (vsiRNAs) biogenesis was conversely increased in plants infected with TYLCV-YN6244-{Delta}V2. Surprisingly, small RNA profiling indicated that 21nt and 22nt rather than 24nt vsiRNAs were predominantly produced in tomato plants infected with either TYLCV-YN6244 or TYLCV-YN6244-{Delta}V2. Furthermore, transcriptome analyses revealed that TYLCV-YN6244 or TYLCV-YN6244-{Delta}V2 infection differentially modulated metabolism and defense-related pathways in tomato, probably underlying distinct viral pathogenicity and disease symptoms induced in plants. Overall, our research not only identified a novel pathogenic TYLCV isolate but also characterized molecular biology and host response in tomato with infectious clones firstly developed, with implications in untangling virus-host interaction for developing novel resistance in crop tomato.

Viral phenotypes such as host and tissue tropism are critical determinants of viral infection and transmission. Inferring viral phenotypes presents unique challenges compared to cellular organisms, as viruses rely entirely on host machinery for replication and survival. Current methods for predicting viral phenotypes mainly rely on viral genomic data, often overlooking host-related information. Here, we evaluated the utility of predicted virus-human protein-protein interactions (PPIs) in inferring diverse viral phenotypes using machine-learning algorithms. For predicting human infectivity, a PPI-based machine learning model outperformed both virus genomic and protein sequence-based models that used large language model embeddings. It also surpassed previous methods that incorporated both viral and host genomic data. The human proteins identified by the model were significantly enriched in functions related to viral infection and immune response. In predicting various phenotypes of human RNA viruses, PPI-based models performed better than virus sequence-based models in forecasting virulence, human transmissibility and transmission routes, while showing comparable performance to genomic sequence-based models in predicting tissue tropism. Finally, we demonstrated that a PPI-based model could distinguish high-risk HPV genotypes from low-risk ones. Proteins associated with high-risk HPV were involved in apoptosis and immune regulation, whereas those linked to low-risk HPV were enriched in telomere maintenance and DNA repair. Collectively, this study is the first to demonstrate the value of predicted virus-human PPIs in inferring viral phenotypes, thereby enhancing our understanding of the molecular mechanisms underlying these phenotypes. It also provides effective tools for risk assessment of emerging viruses, contributing to improved pandemic preparedness.

We developed a convolutional neural network-based machine learning technique to simultaneously analyze the morphology and motility of spirochetal bacteria swimming with continuous cellular deformation. Matching probabilities between experimental images and learned models realizes quantification of cell morphology and association with motility. This method can be applied to diverse transformable cells, offering critical biophysical insights into microbial dynamics.

Background: Large-scale biobanks have advanced genomic and epidemiologic research, but many rely on infrequent biological sampling and limited digital phenotyping. The Yamanashi Multi-omics Cohort (YMoC) was established to support longitudinal assessment of molecular, clinical, and behavioural changes in a screening-defined cohort of adults at elevated metabolic risk without diagnosed diabetes. Methods: YMoC is a longitudinal cohort of 215 adults aged 30-70 years in Yamanashi Prefecture, Japan, who met prespecified glycaemic eligibility criteria at health check-up, including fasting plasma glucose 100-125 mg/dL (5.6-6.9 mmol/L) and HbA1c <6.5%. Participants underwent three in-person visits over six months. Measurements include 75-g oral glucose tolerance testing with serial sampling, clinical biochemistry, anthropometry, liver elastography, and collection of blood, urine, stool, and saliva for multi-omics profiling. Between visits, participants wore a Fitbit Inspire 3 and completed daily app-based questionnaires using the Taohealth app. Current molecular data include genome-wide single nucleotide polymorphism array genotyping and longitudinal plasma proteomics in a subset. Conclusions: YMoC is designed to evaluate within-person molecular and phenotypic trajectories in a screening-defined metabolic-risk cohort. The cohort provides a dense longitudinal resource linking clinical assessments, biospecimens, omics assays, and digital phenotyping, including analyses of insulin-resistance-related markers such as homeostasis model assessment of insulin resistance (HOMA-IR).

Begomoviruses present severe threats to global crop production through complex vector-mediated transmission by the whitefly Bemisia tabaci to host plants such as tomato (Solanum lycopersicum). Unraveling the molecular dialogue between host immune activation and vector non-coding RNA networks is essential for identifying key drivers of virus persistence and transmission. Public host transcriptomic (GSE309527) and vector small RNA (sRNA) sequencing datasets (GSE111343) were processed through a multi-omics harmonization and signal calibration pipeline. Differential expression analysis was performed using empirical Bayes moderated linear models, followed by non-parametric Spearman rank correlation modeling ({rho}) to infer cross-kingdom co-expression dynamics and pathway enrichment profiling across host and vector bio-systems. Harmonized principal component analysis showed clear separation by infection status across host plant and vector cohorts. Differential expression analysis identified 138 significantly altered host genes (69 upregulated, 69 downregulated) and 130 differentially expressed vector sRNAs (65 upregulated, 65 downregulated). Host responses were dominated by significant upregulation of gene-silencing machinery, including Suppressor of Gene Silencing 3 (SGS3; log2 FC = 3.67, q = 7.47 x 10-5), and pathway enrichment in Jasmonate defense (q = 0.0004) and RNA Interference & Silencing (q = 0.0001). Vector sRNAs exhibited targeted dynamic alterations, with pathway enrichment in Salivary Gland Secretion ($q = 0.0030) and Gut Endosymbiont Response (q = 0.0210). Cross-kingdom correlation modeling revealed two distinct, highly anticorrelated regulatory modules (mean |{rho}| = 0.76). Host SGS3 expression strongly correlated with vector sRNA VEC_0080 ({rho} = 0.9762) and virus-derived siRNA Bt-vsiRNA-01 ({rho} = 0.7619). These findings demonstrate a tightly synchronized tripartite molecular crosstalk between host antiviral immunity, viral siRNA accumulation, and vector small RNA remodeling. These cross-kingdom regulatory modules highlight promising targets for dual-action RNA interference strategies aimed at controlling Begomovirus transmission.

CT83 (KK-LC-1) is a cancer-testis antigen originally identified in human lung cancer cells and has recently attracted attention as a potential target for cancer therapy. Although KK-LC-1 orthologs have been identified in up to 160 animal species, a murine homolog had not previously been identified, hindering in vivo analysis of its physiological function. In this study, we identified the mouse homolog of KK-LC-1 and performed a comparative analysis of its properties in humans and mice, together with an investigation of its biological function using gene knockout (KO) mice. The murine Kk-lc-1 gene is located on the X chromosome and, like its human counterpart, contains an N-terminal transmembrane domain. In both humans and mice, KK-LC-1 is expressed specifically in the testis and localizes to the head and tail regions of sperm. Analysis of Kk-lc-1-deficient mice revealed normal spermatogenesis, and both male and female KO mice were fertile. However, sperm from Kk-lc-1-deficient males exhibited reduced motility caused by decreased flexibility of the midpiece and failed to penetrate the oocyte zona pellucida in vitro. This defect was rescued by artificial insemination using epididymal sperm, suggesting that maternal factors in vivo may compensate for reduced sperm motility. Although impaired sperm motility during in vitro fertilization (IVF) was rescued by murine Kk-lc-1, functional rescue by human KK-LC-1 was not observed. These findings indicate that KK-LC-1 contributes to sperm motility but is not essential for fertility. Moreover, species-specific differences in KK-LC-1-mediated regulation of sperm motility suggest functional divergence during evolution. The role of KK-LC-1 in sperm motility should therefore be considered in the clinical development of cancer therapies targeting KK-LC-1.

Compact Cas nucleases offer advantages over the widely used SpCas9 due to their smaller size, which enables more efficient delivery for in vivo applications. Among these, the phage-encoded Cas{Phi}2 (Cas12j2) is highly promising due to its relaxed PAM requirement (5-TTN-3) and compact size (757 aa); however, its translational potential is limited by low editing activity. To enhance the efficacy of Cas{Phi}2, we optimized the previously reported EPICA system, developing EPICA.2, a eukaryotic directed evolution platform to improve nucleases with nearly undetectable activity. EPICA.2 integrates additional yeast evolution rounds to enrich for active variants along with a low background mammalian reporter system that improves detection and selection of enhanced variants. Finally, we set up a long-read sequencing protocol which uses unique molecular identifiers (UMIs) to reduce sequencing errors, enabling accurate identification of the mutation combinations in each evolved variant. Among the most frequent variants, we obtained evoCas{Phi}2, which contains six activity-boosting mutations with a synergistic effect not predictable by rational engineering. Overall, evoCas{Phi}2 showed up to 70-fold increased activity in human cells compared to wild-type and outperformed variants generated through rational approaches, highlighting the potential of EPICA.2 as a powerful strategy to evolve genome editing tools with low native activity.

Giant viruses (GV) are increasingly recognized as important ecosystem regulators. While metagenomics has uncovered extensive GV diversity, the global distributions of individual species and the biogeographic processes driving the pattern remain poorly understood. Here, we reconstructed GV metagenome-assembled genomes (MAGs) from 35 globally distributed deep freshwater lakes spanning five continents, aiming to identify their biogeographic patterns. The resulting 1663 non-redundant MAGs significantly expanded the known freshwater GV diversity, with [~]84% lacking a previously reported species representative. These MAGs were grouped into cosmopolitan and geographically restricted lineages. We identified 27 cosmopolitan GV species spanning multiple viral lineages, including families of Imitervirales, Pimascovirales, and mirusviruses order Styxvirales. The cosmopolitan species were characterized by their larger genomes and expanded gene repertoires of host-interaction functions, which may facilitate interactions with diverse hosts and contribute to their global distributions. The presence of geographically restricted species and the stronger distance-decay in community similarity observed in freshwater than marine ecosystems suggest that physical connectivity between ecosystems is an important factor influencing GV dispersal. We identified 312 and 177 GV MAGs almost exclusively associated with the epilimnion and hypolimnion, respectively. This water-layer preference of individual MAGs was highly consistent across lakes, suggesting conserved vertical partitioning in association with the thermal stratification of the water column. Overall, our findings reveal that GV biogeography in deep freshwater lakes is structured by the combined influence of horizontal dispersal limitation, vertical partitioning, and lineage-specific evolutionary histories.

Influenza virus poses a potential risk of triggering the next global pandemic. In-depth investigation into the mechanisms underlying influenza virus replication and pathogenicity will provide robust support for controlling influenza virus infection. Although post-translational modifications are known to regulate viral infection, the role of lactylation in influenza virus replication remains elusive. In this study, influenza virus ribonucleoprotein complex subunits are found to be lactylated. Specifically, ATAT1 promotes viral polymerase acidic protein (PA) lactylation and enhances viral replication. In contrast, SIRT1 mediates de-lactylation of PA and exerts an inhibitory effect on viral replication. Further investigations reveal lactylation of PA at residues K605 and K609 is essential for viral replication and pathogenicity. Mechanistically, PA K605/609 residues are localized at the interaction interface of the ANP32-mediated polymerase asymmetric dimer; mutation at these residues inhibits polymerase asymmetric dimerization, thereby impairing RNA production during viral genome replication. Collectively, this study uncovers a novel mechanism by which influenza virus hijacks host enzymes to mediate PA lactylation, and expands the molecular regulatory network of influenza virus infection.

A protein whose removal or deletion causes significant disruption or collapse of a protein-protein interaction (PPI) network is referred to as a vulnerable protein. Such proteins may serve as valuable therapeutic or diagnostic targets in disease-associated networks. In this study, two PPI networks were constructed, one for HPV-positive and the other for HPV-negative head and neck squamous cell carcinoma (HNSCC), and the vulnerable proteins of these networks were identified by the node deletion approach. After analyzing the networks, 27 unique vulnerable proteins in HPV-positive and 72 unique vulnerable proteins in HPV-negative HNSCC were identified. Among them, one HPV-positive and seven HPV-negative HNSCC vulnerable proteins were further chosen by integrating multi-omics data. To exploit the vulnerabilities of these proteins, candidate synthetic lethal (SL) partners were predicted whose inhibition may selectively impair tumor survival. Subsequently, drug-gene interaction analysis was performed to identify inhibitors targeting the SL partners of these vulnerable proteins. Notably, in HPV-positive HNSCC, TOP2A, CHEK1, and CHEK2 genes were identified as SL partners of TTN, and their inhibitors were already clinically approved. While in HPV-negative HNSCC, ADA and MMP19 were identified as an SL partner of LMO7; TMEM45B, CDH3, and ELF3 genes were identified as an SL partner of CGN; and ZNF433 was identified as an SL partner of FLNC. However, MMP19, ZNF433, and TMEM45B inhibitors were not reported. Thus, these vulnerable proteins, including their SL partners, provide novel avenues to explore and develop more efficient and precise therapeutic and diagnostic strategies.

Histone proteins, which reside in the nuclei of eukaryotic cells, are involved in diverse cellular processes. The core histone H4 serves as a structural component of the nucleosome. Patients carrying mutations in H4Clustered histone (H4C) genes exhibit a broad spectrum of developmental abnormalities, including short stature, microcephaly, intellectual disability, growth retardation, and digital anomalies. However, the impact of H4 mutations on mammalian embryogenesis remains largely unclear. Here, we demonstrate that histone H4C genes play crucial roles in skeletal development and cortical neurogenesis. We found that mRNAs of the histone H4C gene family are specifically expressed in proliferating progenitor cells in the developing mouse neocortex and in human induced pluripotent stem cell-derived cortical organoids. CRISPR-mediated disruption of H4C3 in mice caused severe defects in skeletal formation and neocortical neurogenesis. Furthermore, overexpression of a mutant form of H4C3 resulted in altered expression of genes associated with cellular migration and motility. Together, these findings suggest that histone H4 plays a critical role in regulating the balance between proliferation and differentiation during mammalian embryonic development, thereby explaining the broad spectrum of patient phenotypes.

Replication-competent coronaviruses carrying fluorescent protein-tagged structural proteins remain scarce. Using the highly attenuated pangolin coronavirus GX_P2V(short_3UTR) as a backbone, we generated GX_P2V-mBJ-N, a recombinant coronavirus in which the bright green fluorescent protein mBaoJin is fused to the nucleocapsid (N) protein. The reporter virus is attenuated and genetically unstable in normal Vero cells but can be amplified to high titers in cells expressing wild-type N, and its fluorescence directly reports N protein expression. Using this authentic-virus platform, we show that high-titer GX_P2V cross-neutralizing antibodies persist in most healthy individuals and that cepharanthine potently blocks viral entry. GX_P2V-mBJ-N thus provides a simple and reliable tool for coronavirus tracing, immune surveillance, and antiviral drug evaluation.

The zebrafish yolk sac (YS) is traditionally viewed as a nutrient reservoir. By reconstructing the complete progression of embryonic neural development via live-cell imaging, we previously uncovered an intriguing involvement of YS after it is innervated by the neurons from the brain, hinting its uncharacterized functional roles beyond nutrient storage. Using transgenic lines and long-term live imaging, we characterized a dynamic neuro-vascular-metabolic interface on the YS surface. We observed that peripheral neural networks expand radially and mature through hierarchical integration, sharing the same structural and dynamical features as those in the brain and spinal cord. To many unexpected, elavl3-positive cells on the YS exhibit collective calcium flashes, suggesting primitive functional communication. Furthermore, we characterized activity-dependent neuronal pruning and stress-induced lipid droplet crystallization as indicators of developmental refinement and homeostatic collapse, respectively. Finally, we identified directional blood flow occurring before the formation of endothelial tubes, indicating a pre-vascular transport mechanism. These findings demonstrate that, empowered by neural innervation, the YS serves as a coordinated developmental hub, facilitating complex crosstalk between neural, vascular, and metabolic systems during early vertebrate embryogenesis.

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

Nucleotide-binding leucine-rich repeat (NLR) immune receptor genes are of significant value in disease resistance breeding and the control of viral diseases. Soybean mosaic virus (SMV) poses a serious threat to soybean production and the Rsv1 locus in soybean cultivar Suweon 97 confers broad-spectrum resistance against SMV strains G1 to G7; however, this locus harbors no fewer than 18 NLR genes, and thus the broad-spectrum antiviral mechanisms underlying the Rsv1 locus remain poorly understood to date. Here, we established a rapid and highly efficient screening system for cloning NLR genes from soybean Rsv1 locus and identified a broad-spectrum antiviral NLR gene 13g184900 from this highly complicated locus. The NLR encoded by 13g184900 can recognize viral P3 protein from all SMV strains (G1-G7) and another potyvirus Bean common mosaic virus (BCMV). The coiled-coil (CC) domain of this NLR directly interacts with viral P3 protein. Additionally, we showed that this NLR originated from wild soybean accession in East China and has been introduced into several soybean cultivars during domestication. Collectively, we developed a high-throughput screening system for identifying NLR genes in soybean and our study provides new mechanistic perspective on how the Rsv1 locus mediates the broad-spectrum resistance to all SMV G1-G7 strains.

Very short RNAs play critical roles in modern biology, and are thought to have been crucial for genome replication during the origin of life. Next-generation sequencing is an essential tool for characterizing pools of small RNAs, but current library preparation methods suffer from strong size and sequence biases. Here we present tinyRNA-seq, an optimized library preparation method designed to minimize length- and sequence-dependent capture bias enabling the sequencing of RNA fragments as short as 2 nucleotides. We use degenerate adaptor regions to reduce ligation sequence bias and facilitate unique molecular identifier (UMI) installation. We benchmarked tinyRNA-seq against commercial kits using a model primordial RNA genome consisting of hundreds of defined oligonucleotides ranging from 2 to 12 nucleotides. tinyRNA-seq reproduced the input RNA distribution without the size and sequence bias of the commercial kits. tinyRNA-seq also enables the detection of de novo oligonucleotide generation, an important process for the origins of life. Applied to biologically derived small RNAs including miRNAs, piRNAs, and cityRNAs, tinyRNA-seq showed significantly lower capture bias and recovered a wider range of sequences than commercial kits. tinyRNA-seq may thus provide a more complete and quantitatively accurate representation of small RNAs from both biological and chemical sources. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/743385v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@95ee64org.highwire.dtl.DTLVardef@155fb06org.highwire.dtl.DTLVardef@1d3665forg.highwire.dtl.DTLVardef@1e61404_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chikungunya virus (CHIKV) causes recurrent epidemics across tropical and subtropical regions globally. In 2025, Guangdong reported mainland China's largest documented CHIKV outbreak, with 23,464 cases across all 21 prefecture-level cities. Integrating epidemiological, genomic, and phylodynamic analyses, we investigated the outbreak's origins, transmission, and viral adaptation. The Guangdong strain belonged to the ECSA-MAL lineage, exhibiting a long internal branch that highlights significant global surveillance gaps. Phylodynamic modeling estimated viral introduction in early April 2025, revealing ~2.5 months of cryptic transmission alongside rising vector densities. Spatial case distribution was moderately associated with human mobility from the epicenters. Globally, phylogenetic analysis identified 33 potential adaptive mutations across nine proteins and 14 epidemic lineages, including validated and 15 novel mutations. Twelve novel mutations occurred in the Asian Urban lineage (AUL), predominantly affecting NSP3. This study underscores the need for enhanced pre-peak surveillance and continuous monitoring of viral adaptation across ecological regions.

Subcellular mRNA localization contributes to local protein synthesis and functional compartmentalization in polarized cells, including neurons and glial cells. Although process-localized mRNAs have been identified by in situ hybridization, reporter-based analyses, and compartment-based transcriptomic approaches, systematic screening of such mRNAs within intact brain tissue while preserving tissue architecture remains challenging. Here, we developed a Xenium-based spatial transcriptomic screening strategy to identify candidate mRNAs localized in neuronal and glial processes in the adult mouse cerebellum. We focused on the molecular layer, which is densely occupied by Purkinje cell dendrites, Bergmann glial radial processes, and granule cell parallel fibers, but contains relatively few cell bodies. Using a Xenium Prime 5K dataset from adult mouse cerebellar sections, we first identified 199 genes whose transcripts were enriched in the molecular layer. By further focusing on DAPI-negative, process-rich regions and reducing contributions from molecular layer cell bodies, we extracted 126 candidate process-localized genes. Comparison with Allen Brain Atlas in situ hybridization data supported molecular layer localization with process-like patterns for 28 of 32 evaluable candidates. Integration with a published adult cerebellar single-nucleus RNA-seq atlas provided information on the possible cellular origins of these candidates. Gene Ontology analysis revealed enrichment of terms related to intracellular transport, cell projection structures, and synaptic function. These findings support the usefulness of a Xenium-based spatial transcriptomic first-screening strategy for identifying candidate process-localized mRNAs in intact brain tissue and provide a resource for studying RNA localization in cerebellar neuronal and glial processes.

Camptothecin derivatives are first-line anticancer drugs used worldwide for the treatment of diverse malignant tumors. However, the biosynthetic pathway of camptothecin has remained elusive for five decades. Here, we fully map its entire biosynthetic route. We discovered five key missing enzymes (OpCAR, OpSDR11, OpCS, OpGH1, and OpSTR) via the combination of MALDI mass spectrometry imaging, single-cell RNA sequencing and co-expression analysis. Meanwhile, we demonstrated a free flavin mononucleotide triggered the non-enzymatic 6-5-6 to 6-6-5 fused-ring skeleton rearrangement, filling the last gap in camptothecin biosynthesis. Finally, we validated this identified pathway and achieved the de novo biosynthesis of camptothecin in Saccharomyces cerevisiae. These discoveries uncover the long-standing mystery underlying camptothecin and pave the way for manufacturing camptothecin and its derivatives through synthetic biology approaches.