Agarwood, renowned as one of the most valuable natural materials in the world of fragrance, is notoriously difficult and time-consuming to obtain, creating a critical bottleneck for its utilization and conservation. Sesquiterpenes are the key constituents of agarwood, with agarospirol recognized as one of its characteristic compounds. Nevertheless, the biosynthesis of agarospirol remains unreported. Here, we conducted a functional screening of terpene synthase (TPS) genes from the agarwood-producing tree Aquilaria yunnanensis. Using a heterologous expression system in Saccharomyces cerevisiae, we characterized 26 TPS genes, identifying 30 structurally diverse sesquiterpene products. Among them, AyTPS1 was identified as an agarospirol-producing sesquiterpene synthase, and the structure of the enzymatically produced agarospirol was unequivocally confirmed by NMR spectroscopy. Furthermore, model-guided mutagenesis of AyTPS1 revealed that residues L283 and G435 markedly influence the distribution of agarospirol and related eudesmane-type sesquiterpenes, suggesting their involvement in shaping the active-site environment for spiro-ring formation and hydroxylated product formation. The elucidation of the AyTPS1 and its catalytic mechanism lays a crucial foundation for the industrial-scale biotechnological production of this valuable sesquiterpene.

β-Caryophyllene, a bicyclic sesquiterpene shows remarkable application potential in agriculture for plant disease prevention, pest control and green pesticide development. However, conventional production methods-relying on plant extraction or chemical synthesis-exhibit inherent limitations that hinder both the achievable yield and the commercial scalability of β-caryophyllene manufacturing. In this study, we mined and identified a highly efficient β-caryophyllene synthase, designated ArarCARS. Through molecular docking and ancestral sequence reconstruction, we screened mutants at key residues and revealed that the F117Y variant significantly enhanced production. Molecular dynamics simulations demonstrated that this mutation improved product formation through enhanced enzyme stability. Based on these results, we engineered a high-titer β-caryophyllene-producing Saccharomyces cerevisiae strain via a series of metabolic engineering strategy. Fed-batch fermentation at low temperatures achieved a final β-caryophyllene titer of 35.4 g/L, representing the highest level documented so far. This work not only provides an effective strategy for β-caryophyllene biosynthesis but also offers engineering insights for the efficient microbial production of other valuable terpenoids.

Sacred lotus is widely used in the agricultural, nutraceutical, and pharmaceutical industries. Terpenes are not only crucial components of sacred lotus essential oil, but also serve as signaling molecules involved in plant-environment interactions. However, the biosynthesis of terpenes in sacred lotus has not yet been reported. Thus, gene-directed heterologous mining and combinatorial biosynthesis methods were used in this study to systematically characterize the function of terpene synthase genes in the sacred lotus. As a result, two monoterpene, 11 sesquiterpene, and three diterpene products were synthesized, and a highly efficient γ-eudesmol synthase was discovered. In addition, a mechanistic study revealed that N314 is the key amino acid responsible for the secondary cyclization that produces γ-eudesmol. In vitro assays demonstrated that γ-eudesmol exhibited substantial insecticidal and antimicrobial activities. Furthermore, de novo biosynthesis of γ-eudesmol was achieved in a yeast chassis through a series of metabolic engineering strategies, reaching a titer of 801.66 mg/L in a shake flask, the highest yield reported to date. The present study uncovered the biosynthesis of terpenes in sacred lotus, as well as successfully synthesized the bioactive compound γ-eudesmol by synthetic biology. This comprehensive strategy can be readily adapted for investigation and the production of other valuable plant-derived natural products.

Plants produce a large array of natural products which play important roles in flavours, fragrances and medicines. However, some high-value plant intermediate metabolites cannot be directly extracted from plants. The tulip tree (Liriodendron chinense) in the Magnoliaceae family is rich in sesquiterpenes. Upon characterizing the functions of 11 Liriodendron chinense terpene synthases, we discovered that LcTPS3 could produce high yields of (+)-germacrene A, which was shown to be a central scaffold in sesquiterpene biosynthesis. This compound can be completely transformed into β-elemene at high temperature, a broad-spectrum antitumor drug widely used in clinical treatment. By expressing LcTPS3 in a precursor-providing Saccharomyces cerevisiae chassis and with the aid of metabolic engineering, the fermentation yield of (+)-germacrene A has been achieved at 14.71 g/L. Site-directed mutagenesis experiments and molecular dynamics simulations revealed that the A280V suppresses the cyclization of substrate by influencing the conformation of the enzyme-substrate. The Y282L facilitates secondary cyclization to produce α-guaiene by shortening the distance between the catalytic residue Y531 and the substrate. These insights underscore the high plasticity of LcTPS3 and suggest that its targeted engineering could unlock the synthesis of a wider array of valuable sesquiterpenes.

Glycosylation plays an important role in the structural diversification of plant natural products. The identification of efficient glycosyltransferases is also a crucial step for the biosynthesis of valuable glycoside products. However, functional characterization of glycosyltransferases (GTs) from an extensive plant gene list is labour-intensive and challenging. Salidroside is a bioactive component derived from plants, widely utilized in the fields of food and medicine. Here, through transcriptome analysis and structure-based virtual screening, we identified two GTs that participated in the biosynthesis of salidroside from a rarely studied herbaceous plant, Astilbe chinensis. Ach15909 was found to possess high catalytic activity as evidenced by the determination of its catalytic parameters. The key residues that determine its catalytic activity were further determined. Additionally, Ach15909 shows a preference for substrates with a volume of <150 Å3, and replacing the interdomain linker region located between the N- and C-terminal domains of Ach15909 allows it to accept substrates that were previously not catalyzable. Overall, the structure-based virtual screening approach showed high efficiency and cost-effectiveness; the successful identification of GTs in salidroside glycosylation sheds light on uncovering additional plant biosynthesis enzymes in the forthcoming research.

Pleuromutilin, a tricyclic diterpene compound with significant inhibitory activity against gram-positive bacteria and mycoplasmas, serves as a precursor for various veterinary and human medicines. Previous efforts have primarily focused on strain screening and fermentation process optimization to enhance pleuromutilin production in native pleuromutilin-producing strains, with the absence of genetic engineering strategies. In this study, we performed whole-genome sequencing of the pleuromutilin-producing strain Clitopilus passeckerianus T6 to identify the biosynthetic genes related to pleuromutilin production. Transcriptomic and metabolomic data were collected during the fermentation of C. passeckerianus T6, and gene transcription and metabolite accumulation in the pleuromutilin biosynthetic pathway were analyzed to identify the rate-limiting steps in pleuromutilin biosynthesis. Overexpression of the key genes ple-ggpps and ple-cyc increased pleuromutilin production by 50%, achieving a titer of 6.9 g/L. This study is the first to employ metabolic engineering to enhance pleuromutilin production in a native strain, providing a strategy for efficient pleuromutilin production.

In clinical practice, pirarubicin (THP) is a widely used triple-negative breast cancer (TNBC) agent. It has been found that circular RNAs (circRNAs) are involved in cancer treatment and progression. However, the biological function of circRNAs in TNBC and the relationship between THP and circRNAs remain poorly studied. circSTIL (hsa_circ_0000069) was screened and validated by bioinformatics analysis, demonstrating that it was highly expressed in TNBC cell lines and plasma exosomes, and correlated with a poor prognosis of patients. The expression level of circSTIL in patients’ plasma exosomes has potential diagnostic value in distinguishing TNBC from non-TNBC. In vitro studies confirmed that overexpression of circSTIL promotes the proliferation, migration, and invasion of MDA-MB-231 cells whereas silicification of circSTIL shows the reverse effect. Also, circSTIL mediates THP inhibiting the malignant phenotype of MDA-MB-231 cells. The above results suggested that circSTIL is a possible biomarker for the diagnosis, treatment, and prognosis of TNBC.

Astilbe chinensis, a perennial ornamental plant in the Saxifragaceae family, is recognized for its medicinal properties due to its diverse secondary metabolites. Here, we generate a chromosome-level genome assembly of A. chinensis. Our analysis provides compelling evidence that A. chinensis experienced a whole-genome triplication event, which preceded the diversification of the Saxifragaceae family. Furthermore, we identify a biosynthetic gene cluster that includes nine terpene synthase (TPS) genes. Among these, the gene AcTPS2 encodes a eudesma-5,7-diene synthase, and the product is confirmed using nuclear magnetic resonance spectroscopy. A synteny analysis of this gene cluster across various representative plant species reveals variations in the number, sequence, and function of TPS genes, indicating that neo-functionalization of these TPS genes likely occurred after speciation. Collectively, the genome sequence of A. chinensis lays the foundation for genetic and evolutionary studies of the Saxifragaceae family and provides insights into terpene synthases discovery.

Plant terpenoids are a vital source of natural products, yet their discovery is often hindered by low abundance or cryptic expression. The integration of genome mining and synthetic biology has emerged as a transformative solution, enabling systematic discovery and characterization of novel terpenoid scaffolds across all major terpenoid classes, including triterpenes, sesterterpenes, diterpenes, and sesquiterpenes. This review highlights key advances achieved through genome-wide functional characterization of terpene synthases, combinatorial biosynthesis approaches, and scalable production systems for bioactivity evaluation. These integrated strategies have proven particularly valuable for uncovering hidden terpenoid diversity and studying naturally rare terpenoids in plants, significantly expanding the chemical space available for pharmaceutical development and drug discovery.

The global threat of MRSA demands innovative anti-virulence strategies. Caseinolytic peptidase P (ClpP), a central virulence regulator in MRSA, represents an attractive yet underexploited target. Here, we developed a discovery platform integrating self-resistance gene-guided genome mining with dual functional screening, combining fluorometric-based assay and counter-screening against ADEP-induced ClpP activation. This led to the discovery of streptoclipamides A–G, novel hybrid polyketide-nonribosomal peptide ClpP inhibitors from str BGC, validated via heterologous expression and gene knockout. Structure–activity relationship studies enabled by engineered analogues identified key pharmacophores. Streptoclipamide A potently inhibits ClpP (IC 50  = 480 nM) by engaging Thr72 via its C-21 hydroxyl group, confirmed by biophysics and self-resistance-conferring T72P mutation. Streptoclipamide A suppressed MRSA virulence in vitro by reducing critical toxin production, including α-hemolysin, and demonstrated protection in Galleria mellonella and murine pneumonia models. This work expands chemical diversity of ClpP-targeting agents, and establishes a genome mining-driven platform for discovering new therapeutics against antibiotic-resistant pathogens.

Ent-abietane diterpenoids constitute a class of terpenes with a C20 carbon skeleton that underlie a wide range of biological activities. Ent-abietane diterpenoids, enantiomeric to the abietane counterparts, represent a family of diterpenoid natural products characterized by their distinct 6/6/6 tricyclic carbocyclic skeletons with exceptional structural complexity. An increasing number of these ent-abietane diterpenoids have recently been identified, constituting a well-defined group of naturally occurring compounds. This review provides a comprehensive summary of the natural sources, chemical structures, biological profiles and total synthesis of these ent-abietane diterpenoids from 2016 to early 2025.

Conserved serine phosphorylation regulates histone deacetylase activity in Arabidopsis and humans

Pneumocandin B0 (PB0) is a lipohexapeptide synthesized by Glarea lozoyensis and serves as the precursor for the widely used antifungal drug caspofungin acetate (Cancidas®). However, the low titer of PB0 results in fermentation and purification costs during caspofungin production, limiting its widespread clinical application. Here, we engineered an efficient PB0-producing strain of G. lozoyensis by systems metabolic engineering strategies, including multi-omics analysis and multilevel metabolic engineering. We overexpressed four rate-limiting enzymes: thioesterase GLHYD, two cytochrome P450s GLP450s, and chorismate synthase GLCS; knocked out two competing pathways responsible for producing 6-methylsalicylic acid and pyranidine E; and overexpressed the global transcriptional activator GLHYP. As a result, the PB0 titer increased by 108.7% to 2.63 g/L at the shake-flask level through combinatorial strategies. Our study provides valuable insights into achieving high-level production of PB0 and offers general guidance for developing efficient fungal cell factories to produce polyketide synthase-non-ribosomal peptide synthetase hybrid metabolites.

Plants contain a vast array of natural products yet to be discovered, particularly those minor bioactive constituents. Identification of these constituents requires a significant amount of plant material, presenting considerable technical challenges. Mugwort (Artemisia argyi) is a widely recognized insect repellent herb, particularly renowned for its extensive usage during the Dragon Boat Festival in China, but the specific constituent responsible for its repellent activity remains unknown. Here, we employed a gene-directed in vitro mining approach to characterize mugwort terpene synthases (TPSs) systematically in a yeast expression system. Based on the establishment of "Terpene synthase-standard library", we have successfully identified 54 terpene products, including a novel compound designated as cyclosantalol. Through activity screening, we have identified that (+)-intermedeol, which presents in trace amount in plants, exhibits significant repellent activity against mosquitoes and ticks. After establishing its safety and efficacy, we then achieved its biosynthetic production in a yeast chassis, with an initial yield of 2.34 g/L. The methodology employed in this study not only identified a highly effective, safe, and commercially viable insect repellent derived from mugwort but also holds promise for uncovering and producing other valuable plant natural products in future research endeavors.

Many studies have investigated the transfer of skills between laparoscopic and robot-assisted surgery (RAS). These studies have considered time, error, and clinical outcomes in the assessment of skill transfer. However, little is known about the specific operations of the surgeon. Clutch control use is an important skill in RAS. Therefore, the present study aimed to propose a novel objective algorithm based on computer vision that can automatically evaluate a surgeon’s clutch use. Additionally, the study aimed to evaluate the correlation between clutch metrics and surgical skill on different surgical robot platforms. The robotic surgery training center of Wuhan University trained 30 laparoscopic surgeons as the study group between 2023 and 2024. Laparoscopic surgeons were trained by combining robotic simulator exercises and RAS animal experiments. During the training, video and hand movement data were collected. Hand movements identified by a skin-color model were combined with labeling information to classify clutch use. The metrics were validated on different robotic platforms (dv-Trainer, EDGE MP1000, Toumai™ MT1000, and DaVinci Xi system) and among surgeons with different surgical skill levels. On the robotic simulator, clutch accuracy in the expert group was significantly higher than in the study group for all tasks. No significant differences were observed in the number of clutches between the expert and study groups. In the RAS experiment, the number of clutches decreased significantly for both study and expert groups. The accuracy was maintained at a high level in the expert group but decreased rapidly in the study group. We proposed a new objective assessment of surgical skills, clutch use metrics, in cross-platform RAS. Additionally, we verified that the metrics significantly correlated with the surgical skill levels of the surgeons.

Passion fruit (Passiflora edulis) possesses a complex aroma and is widely grown in tropical and subtropical areas. Here, we conducted the de novo assembly, annotation, and comparison of PPF (P. edulis Sims) and YPF (P. edulis f. flavicarpa) reference genomes using PacBio, Illumina, and Hi-C technologies. Notably, we discovered evidence of recent whole-genome duplication events in P. edulis genomes. Comparative analysis revealed 7.6∼8.1 million single nucleotide polymorphisms, 1 million insertions/deletions, and over 142 Mb presence/absence variations among different P. edulis genomes. During the ripening of yellow passion fruit, metabolites related to flavor, aroma, and color were substantially accumulated or changed. Through joint analysis of genomic variations, differentially expressed genes, and accumulated metabolites, we explored candidate genes associated with flavor, aroma, and color distinctions. Flavonoid biosynthesis pathways, anthocyanin biosynthesis pathways, and related metabolites are pivotal factors affecting the coloration of passion fruit, and terpenoid metabolites accumulated more in PPF. Finally, by heterologous expression in yeast (Saccharomyces cerevisiae), we functionally characterized 12 terpene synthases. Our findings revealed that certain TPS homologs in both YPF and PPF varieties produce identical terpene products, while others yield distinct compounds or even lose their functionality. These discoveries revealed the genetic and metabolic basis of unique characteristics in aroma and flavor between the 2 passion fruit varieties. This study provides resources for better understanding the genome architecture and accelerating genetic improvement of passion fruits.

Osteoblasts and osteoclasts play an important role in maintaining the structural integrity of bone tissue, in which osteoclasts degrade bone structure and osteoblasts restore bone tissue. The imbalance of osteoblast and osteoclast function can lead to many bone-related diseases, such as osteoporosis and inflammatory osteolysis. The drug that can both promote bone formation and inhibit bone loss will be able to treat those diseases. In this study, it was found that LMK-235, an selective HDAC4/5 inhibitor, inhibited the differentiation and maturation of osteoclasts by regulating NF-κB and p-Smad2/3 signaling pathways via inhibition of HDAC4. At the same time, we found that LMK-235 promoted osteoblast mineralization by upregulating Runx2 expression via inhibition of HDAC4. In vivo, LMK-235 was able to alleviate lipopolysaccharide (LPS)-induced calvarial osteolysis and promote the repair of bone defects. Taken together, LMK-235 suppresses osteoclast differentiation and promotes osteoblast formation by inhibiting HDAC4. This may provide a valuable treatment for bone diseases caused by abnormal osteoclast bone resorption and osteoblast bone regeneration.

Glycosylation plays a very important role in plant secondary metabolic modifications. Neodiosmin, identified as diosmetin-7-O-neohesperidoside, not only acts to mitigate bitterness and enhance the flavor of food but also serves as a pivotal metabolite that reinforces plant immunity. Investigating its biosynthetic pathway in plants is crucial for optimizing fruit quality and fortifying plant immune responses. In this study, through analysis of transcriptomic data from Astilbe chinensis, we identified two novel uridine diphosphate (UDP)-glycosyltransferases (UGTs): Ach14791 (AcUGT73C18), responsible for flavonoid 7-O-glycosylation and Ach15849 (AcUGT79B37), involved in flavonoid-7-O-glucoside-2″-O-rhamnosylation. By delving into enzymatic properties and catalytic promiscuity, we developed a biosynthesis route of neodiosmin by establishing a one-pot enzyme-catalyzed cascade reaction. Simultaneously, lonicerin and rhoifolin were also successfully synthesized using the same one-pot dual-enzyme catalytic reaction. Taken together, our findings not only identified two novel UGTs involved in neodiosmin biosynthesis but also provided important biocatalytic components for the microorganism-based biosynthesis of flavonoid-7-O-disaccharide compounds.

Introduction Nucleosomes harboring specific histone variants show distinct chromatin localization patterns and regulatory functions, thereby playing crucial roles in epigenetic regulation. Compared to the well-understood variants of H2A and H3, the study about H2B variants is emerging. Deciphering the roles and regulatory mechanisms of H2B variants in plants will provide more knowledges about epigenetic regulations in plant biology. Objectives Using the model plant Arabidopsis thaliana as the research subject, we systematically analyzed histone H2B variants, four short N-terminal histone H2B variants (snH2Bs) were identified. The genomic distribution characteristics of these snH2Bs, their impact on plant growth, and the potential regulatory mechanisms were studied. Methods By integrating whole-genome chromatin immunoprecipitation sequencing (ChIP-seq) and fluorescence microscopy localization analysis, the distribution of snH2Bs across the genome was identified. Single, double, and triple knock-out mutants were constructed using CRISPR-Cas9 to further explore the functions of snH2Bs in the growth process of Arabidopsis thaliana, the possible mechanisms were also discussed. Results These snH2B variants are preferentially expressed in reproductive tissues and are detected in the nuclei of pollen grains. Further genome-wide profiling indicates that the snH2Bs distribute at active chromatin regions and are positively correlated with gene expression. By creating knock-out single, double, and triple mutants for these snH2Bs, we demonstrate that H2B.5 influences vegetative to reproductive transition. We also show that H2B.5 is required for proper accumulation of H3 lysine 9 acetylation and H2B mono-ubiquitination. Conclusion Overall, our study not only provide insights into the functions and chromatin characteristics of plant snH2Bs, but also supplies examples that illustrate the interplay between histone variants and histone modification. These findings contribute to the understanding of the fundamental principles of epigenetic regulation in eukaryotes and also highlight potential targets for crop improvement.