Yousong Ding

Native bacteria are strains isolated from the host and often require greater development and optimization of genetic manipulation tools than lab-adapted strains. We have previously shown that the native murine Escherichia coli strain EcAZ-1, which colonizes the conventional mouse gut long term, is genetically tractable, and the engineered strain can elicit physiological effects while residing in the mouse gut. Given the interest in using EcAZ-1 and other native E. coli as a chassis for heterologous gene expression in the mouse gut, researchers need more efficient tools to rapidly and reliably introduce heterologous gene expression into the strain. Here, we demonstrate that a gene expression cassette can be integrated into the EcAZ-1 genome with high efficiency using the site-specific Tn7 transposon. Furthermore, we demonstrate that EcAZ-1 modified to express sfGFP using this method stably engrafts in the gut of conventional mouse. Taken together, Tn7-mediated integration is an effective method for stably introducing genetic cassettes into the native murine strain EcAZ-1 for expression in the mouse gut.

Elucidating the biological origins and biosynthetic pathways of marine natural products remains a major challenge, particularly for compounds derived from microbial consortia. Here, we identify the marine cyanobacterial genus Dapis as the source of the potent anticancer natural product dolastatin 15 using genome-resolved metagenomics. Reconstruction of two complete and two near-complete genomes provides genome-resolved characterization of the genus Dapis , revealing extensive biosynthetic potential. Genome mining identified the dolastatin 15 biosynthetic gene cluster. Subsequent analyses reconstructed its pathway and uncovered a noncanonical five-domain nonribosomal peptide synthetase module containing an embedded 2-keto acid–activating domain, revealing an unexpected architectural solution for incorporating 2-keto acid-derived hydroxy acid building blocks. Comparative and evolutionary analyses support a model in which this architecture may have arisen through recruitment of keto acid-activating domains and reduction of canonical adenylation domain features. We further biochemically and structurally characterize an O -methyltransferase that catalyzes formation of the characteristic methoxy pyrrolinone moiety, thereby validating a key terminal tailoring step in the proposed biosynthetic pathway. Together, these findings define the biosynthetic logic of dolastatin 15 and expand understanding of substrate activation and assembly-line diversification in nonribosomal peptide biosynthesis.

Marine-derived fungi are a rich source of structurally diverse secondary metabolites with unique biological activities. Guided by an integrated GNPS molecular networking strategy, chemical investigation of the culture extract of the marine-derived fungus Aspergillus ochraceopetaliformis FM451 led to the isolation of four bis-indolyl alkaloids (1–4). Their structures were elucidated as asterriquinol G (1), asterriquinone K (2), asterriquinone C-1 (3), and asterriquinol E (4) based on comprehensive NMR spectroscopic analyses and HRESIMS data. The enantiomers of asterriquinol G (1a and 1b) were further separated by semi-preparative chiral HPLC, and their absolute configurations were assigned by comparison of the experimental and calculated specific rotations. In addition, the absolute configuration of asterriquinone K (2) was assigned as 11″R based on its specific rotation. Although the planar structures of compounds 1 and 2 had previously been reported, 11″R-asterriquinol G (1a), 11″S-asterriquinol G (1b), and 11″R-asterriquinone K (2) represent the first complete stereochemical characterization of these bis-indolyl alkaloids. All isolated compounds were evaluated for their effects on the growth and biofilm-associated phenotypes of Candida albicans. Although their effects on fungal growth were limited, compound 1a produced the greatest reduction in surface-associated biofilm biomass among the tested compounds, reducing crystal violet-stained biomass to approximately 50% of the control level at 16 μg/mL. Its enantiomer 1b was associated with more pronounced alterations in residual biofilm morphology, with fewer apparent hyphal structures than those observed following treatment with 1a at comparable concentrations. These findings provide preliminary evidence that the stereochemistry of asterriquinol G may differentially influence its biofilm-associated effects on C. albicans and provide a basis for further stereochemistry-oriented studies of bis-indolyl alkaloids from A. ochraceopetaliformis FM451.

Polyketides are a major class of bioactive natural products whose activities are often determined by the stereochemistry of β-hydroxyl groups. In type I polyketide synthases (PKSs), ketoreductase (KR) domains establish these stereocenters, making accurate prediction of KR stereochemistry important for natural product discovery and PKS engineering. Existing rule-based methods rely on a limited set of sequence motifs and often perform poorly across phylogenetically diverse taxa. Here, we present KRstereo, a machine learning framework that predicts KR stereochemistry directly from sequence. Analysis of β-modular KR domains from MIBiG 3.1 identified informative sequence features beyond canonical motifs, motivating the use of protein language model embeddings. KRstereo achieved accuracies of up to 95.7% across taxa and 93.0% for non-Streptomyces KRs, consistently outperforming existing rule-based approaches. Validation using newly characterized KR domains from MIBiG 4.0 confirmed strong generalizability, including cases misclassified by current methods. Application of KRstereo to 20,840 β-modular KR domains from antiSMASH enabled large-scale stereochemical annotation of previously uncharacterized PKS systems. By linking sequence to stereochemical function, KRstereo improves reconstruction of polyketide structures from biosynthetic gene clusters and facilitates stereochemistry-aware genome mining and engineering of PKS assembly lines.

Marine cyanobacteria are a rich source of diverse bioactive natural products, targeting proteins involved in many diseases. Here, we combined metagenomic analysis to enhance the structure elucidation process of two new cyclodepsipeptides named dapalides D (1) and E (2) from a collection of a cyanobacterial mat containing multiple Dapis species from Guam. Dapalides D/E are composed of 11 amino acids, including multiple identical units with different configurations. Enantioselective amino acid identification of the acid hydrolyzate established the identity of amino acids, including the configuration of α/β-stereogenic centers. Identification and analysis of the dapalides D/E biosynthetic gene cluster from a metagenome-assembled genome aided the elucidation of α-configuration and establishment of the order of individual building blocks, collectively revealing the total structure. Phylogenomic analysis indicates that the dapalides D/E producer belongs to Dapis sp. (Dapis sp. VPG23–80 MAG-2), which shares a 95.2% average nucleotide identity with Dapis sp. VPG23–80 MAG-1, the producer of dapalides A–C that cooccurs in the same assemblage. Dapalide D (1) showed moderate growth inhibitory activity against various cancer cell lines. This work expands the dapalide structure class and further highlights the use of combined chemical and metagenomic analyses for natural product structure elucidation.

An investigation of the Hawaiian marine-derived fungus Fusarium sp. strain FM701 led to the isolation of two new pyridinium-containing fusaric acid dimers (1, 2), fusaric acid (3), and two known fusaric acid derivatives (4, 5), beauvericin (6), beauvericin J (7), and desferricrocin (8). Structures of 1 and 2 were elucidated by NMR spectroscopy, GIAO NMR calculations, HRESIMS, and electronic circular dichroism analyses. Genome mining revealed putative biosynthetic gene clusters for fusaric acid, beauvericin, and desferricrocin. Chemical synthesis of 9,10-dehydrofusaric acid (4) and racemic fusarinolic acid (5), combined with feeding experiments, supported their plausible biosynthetic relationship and potential roles as biosynthetic building blocks of dimeric metabolites 1 and 2. Compounds 1, 2, 6, and 7 exhibited potent antibacterial activity against Gram-positive bacteria, and all compounds (1–8) showed 2- to 4-fold enhanced activity when combined with a subinhibitory concentration of chloramphenicol. Beauvericin (6) also displayed strong antiproliferative activity against human breast (MCF-7) and prostate (DU145) cancer cell lines, with low micromolar IC₅₀ values. Together, these findings expand the chemical diversity of fusaric acid metabolites and the biological potential of marine fungal metabolites.

Aldoximes are amino acid-derived metabolites that serve as precursors of auxins and modulate phenylpropanoid production in Arabidopsis. However, the enzymes responsible for aldoxime production in Solanaceae remain unknown. Here, we report the identification of aldoxime-producing enzymes in tomato (Solanum lycopersicum) and examine how altered aldoxime production affects auxin production and phenylpropanoid metabolism. Through homology-based analysis, we identified five putative CYP79 homologs in tomato, among which SlCYP79DB32 and SlCYP79DB52 exhibited aldoxime-producing activity toward multiple amino acids, including phenylalanine and tryptophan. SlCYP79DB32 and SlCYP79DB52 converted phenylalanine into phenylacetaldoxime (PAOx), whereas only SlCYP79DB52 converted tryptophan into indole-3-acetaldoxime (IAOx). Stable isotope-labeled feeding experiments revealed that IAOx and PAOx can be converted to the auxins indole-3-acetic acid (IAA) and phenylacetic acid (PAA), respectively. Consistently, tomato plants engineered to overproduce IAOx and PAOx accumulated elevated levels of IAA and PAA. These plants also accumulated lower levels of phenylpropanoids. In Brassicaceae plants such as Arabidopsis and Camelina, aldoxime accumulation represses phenylpropanoid production by promoting degradation of phenylalanine ammonia-lyase (PAL). However, aldoxime accumulation did not reduce PAL activity in tomato, suggesting an alternative mechanism in this species. Transcriptome analysis revealed extensive transcriptional reprogramming in aldoxime-overaccumulating tomato plants, including upregulation of stress- and defense-related genes. Despite the observed reduction in phenylpropanoid levels, transcript levels of most phenylpropanoid biosynthetic genes were not decreased, suggesting possible post-transcriptional regulation of this repression. Together, our findings demonstrate that aldoximes can serve as intermediates in auxin biosynthesis in tomato and reveal that aldoxime-mediated repression of phenylpropanoid metabolism extends beyond Brassicaceae.

Nonheme Fe(II)/2-oxoglutarate (Fe/2-OG) dioxygenases carry out a broad range of oxidative reactions, yet α-hydroxylation of glycyl residues remains exceedingly rare. Here, we demonstrate that the Fe/2-OG enzyme MysH from Nostoc linckia performs this unusual chemistry during mycosporine-like amino acid biosynthesis, converting mono- and disubstituted precursors into palythines and revealing unexpected substrate tolerance. Kinetic isotope effects, detection of a transient hydroxylated intermediate, and glyoxylate byproduct formation support an α-hydroxylation-initiated mechanism. High-resolution crystal structures, complemented by molecular docking, molecular dynamics simulations, and site-directed mutagenesis, define an active-site architecture that positions the glycyl substrate in a near-transition-state geometry. Hybrid QM/MM calculations reveal a low-barrier hydrogen-atom-transfer step followed by hydroxyl rebound and implicate a conserved Trp125 in an electron-transfer network that lowers the activation barrier. Together, these findings establish a mechanistic framework for protein-directed α-glycine C–H activation by nonheme iron enzymes and provide a blueprint for engineering Fe/2-OG dioxygenases to expand the chemical diversity of mycosporines and related natural products.

Biaryl motifs are fundamental structural elements in many pharmaceuticals, agrochemicals, and advanced materials. Traditional synthetic approaches for biaryl bond formation often require harsh conditions, costly catalysts, and pre-functionalized starting materials, which limit their efficiency, sustainability, and substrate scope. Enzymatic catalysis offers a greener alternative. However, biocatalysts capable of directly coupling halogenated biaryl compounds remain largely underexplored. Here, we report the functional characterization of the marine-derived cytochrome P450 enzyme Bmp7, which catalyzes the formation of halogenated biaryls. We first characterized the product profile of recombinant Bmp7 using its native substrate 2,4-dibromophenol (1) and confirmed the dominant ortho-ortho C-C homocoupled product as MC21-A. Screening a halogenated aromatic substrate library revealed that Bmp7 binds and catalyzes the coupling of 17 halogenated phenols, as evidenced by spectral shift assays, LC-HRMS, HRMS/MS and GC-MS analyses. Two homocoupled products were structurally confirmed by NMR analysis to possess ortho-ortho C-C linkages. In addition to efficient homocoupling, Bmp7 catalyzed heterocoupling reactions between substrate 1 and 16 other substrates, producing mixtures of homocoupled and heterocoupled halogenated biphenols. X-ray crystallography revealed the binding of two substrate 1 molecules within the active site, while DFT calculations supported a single-radical reaction mechanism, shedding light on the mechanistic basis of the coupling reaction. Together, these findings lay the groundwork for these findings establish a foundation for future efforts in enzyme engineering and the development of biocatalytic strategies for synthetic applications.