Zixin Deng

Bacillus subtilis is widely used as a probiotic to mitigate alcohol-induced gut-liver injury, yet its therapeutic potential is limited by poor survival under gastrointestinal and ethanol stress, often necessitating encapsulation for viable delivery. Here, we isolated food-borne B. subtilis strains carrying DNA phosphorothioation (PT)—a DNA backbone modification in which sulfur replaces a non-bridging oxygen. The PT strain exhibited markedly enhanced tolerance to multiple stresses compared with the PT-deficient mutant, an effect we attribute to PT-mediated neutralization of reactive oxygen species (ROS) generated by ethanol and gastrointestinal conditions. In an acute mouse model of alcohol-induced injury, the PT strain showed a favorable safety profile and superior efficacy in reducing oxidative damage. In a chronic weaned piglet model, the PT strain achieved higher gastrointestinal survival and conferred stronger hepato-intestinal protection, reversing gut microbiota dysbiosis, preserving mucosal barrier integrity, and attenuating hepatic steatosis. Mechanistically, B. subtilis converts luminal taurochenodeoxycholic acid (TCDCA) into the cytoprotective bile acid tauroursodeoxycholic acid (TUDCA), which activates host TGR5 signaling to reinforce the intestinal barrier. This barrier fortification blocks lipopolysaccharide (LPS) translocation into the portal circulation, thereby suppressing the hepatic IFN-γ/JAK-STAT cytokine cascade and ameliorating liver damage. Our findings establish DNA phosphorothioation as a built-in stress shield that enables robust probiotic delivery in the alcohol-exposed gastrointestinal tract, providing a strategy for engineering resilient live biotherapeutics against gut-liver axis disorders.

All eukaryotes, including yeast, plants, animals and humans, possess linear chromosomes. The conserved eukaryotic telomere-telomerase systems, originated and evolved over 1 billion years, protect the chromosomal ends and regulate critical physiological functions through complex networks. In this study, we replace the endogenous eukaryotic telomeres in the single-chromosome yeast Saccharomyces cerevisiae with the prokaryotic telomere system TelN/tos from the Escherichia phage N15, which forms a closed hairpin structure, by interrupting the MRX/Sae2 pathway. The prokaryotic telomeres effectively protect linear chromosomal ends and prevent genetic instability. Through adaptive evolution, we identify yeast strains harboring additional mutations (TEL1 and CYR1) that restore functional MRX/Sae2 activity, thereby improving host fitness and meiotic capacity. Interestingly, the two-associated TelN/tos telomeres position deeper into chromosomes and exhibit increased interactions with their adjacent regions. The successful replacement of a complex eukaryotic chromosomal telomere with a simple bacteriophage system demonstrates functional equivalence between these divergent systems, implying possible natural origins of such stochasticity (e.g., horizontal gene transfers). Furthermore, these engineered strains facilitate development of a tos-YAC system that enabled iterative assembly and stable maintenance of megabase-level heterogeneous DNA (1.23-2.77 Mb), providing a robust platform for large-scale DNA manipulation.

The genus Fusobacterium encompasses significant pathogens implicated in diseases spanning from infections to cancer. However, taxonomic ambiguities persist within the genus, particularly concerning Fusobacterium nucleatum (sensu lato). Through genus-wide average nucleotide identity (ANI) and phylogenetic analyses of 540 Fusobacterium genomes, we identify an ANI gap (93.38%−93.89%) for species delineation, leading to comprehensive taxonomic revisions that resolve these ambiguities. We further establish gyrB and rpoB as high-resolution taxonomic markers with phylogenies consistently supporting the revised taxonomy. Leveraging these markers, we develop B&B, a general strategy for precise species identification without whole-genome sequencing, and validate its accuracy in clinically relevant strains. Integrating the revised taxonomy with genomic/metagenomic toolkits demonstrate broad utilities, reinterpreting key colorectal cancer-associated species. This work establishes a unified taxonomic framework and enables standardised species classification for Fusobacterium isolates and microbiomes, highlighting the genetic divergence among Fusobacterium species and providing the taxonomic precision essential for advancing Fusobacterium-related research.

Gastric cancer (GC) is one of the most common malignancies worldwide and it is the third leading cause of cancer-related death in China. While Helicobacter pylori is a known GC pathogen, its abundance declines in tumours and the role of other bacteria in GC metastasis remains unclear. We aim to investigate the mechanisms of other bacteria influencing GC progression and metastasis. Integrated intratumoural microbiome-metabolome analysis identified GC-associated microbes and metabolites. We then demonstrated the pro-metastatic role of Acinetobacter baumannii (A. baumannii, Ab) and its metabolite nicotinic acid (NA) using genetic, molecular and in vivo approaches. The abundance of A. baumannii was significantly increased in GC tissues, correlating with advanced tumour stage and intratumoural NA levels. Fluorescence in situ hybridisation confirmed its colonisation in GC tumours. In co-culture systems, A. baumannii increased NA levels, enhancing nicotinamide adenine dinucleotide (NAD) metabolism and increasing 1-Methylnicotinamide accumulation in tumour cells. Mutagenesis of the bacterial NA synthase gene pncA confirmed that A. baumannii excreted an NA-dependent pro-metastasis effect. Mechanically, A. baumannii promotes GC metastasis by reprogramming tumour cell glucose metabolism, reducing oxidative phosphorylation while enhancing glycolysis and activating the hypoxia-inducible factor-1 pathway in GC cells through metabolites both in vivo and in vitro. This study elucidates the role of A. baumannii in enhancing NAD metabolism in GC cells through NA synthesis, consequently promoting GC metastasis. These findings establish a microbiota-metabolism axis as a mechanistic foundation for developing targeted therapeutic strategies against GC metastasis.

Steroids are among the most valuable and widely used pharmaceuticals. The cholesterol side-chain cleavage enzyme (P450scc) is critical for steroid metabolism and hormone biosynthesis. While mammalian eukaryotic P450scc enzymes are well-characterized, bacterial counterparts remain underexplored despite their industrial promise and potential contributions to bacterial steroid catabolism. Here, we identify a series of CYP204 family P450 enzymes, widely distributed across diverse steroid-degrading bacterial species, that catalyze the side-chain cleavage of cholesterol, phytosterol, and cholestenone to produce pregnenolone and progesterone. Unlike mammalian enzymes, which exhibit strict cholesterol specificity, bacterial P450scc enzymes display relaxed substrate specificity, preferentially converting cholestenone to progesterone—a key precursor in steroid drug semi-synthesis. Structural and mechanistic analyses demonstrate that CYP204 enzymes employ a flexible, dual-regioselective C–H activation mechanism distinct from the sequential hydroxylation of mammalian P450scc enzymes. Iterative saturation mutagenesis identified critical residues for side-chain cleavage, improving catalytic efficiency up to 6.5-fold, and computational analyses clarified sequence–function relationships. This finding of bacterial P450scc enzymes not only underscores their potential function in bacterial steroid catabolism but also lays a foundation for promising biocatalytic strategies for pregnenolone and progesterone synthesis.

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.

MRNA vaccines, featured by incorporated pseudouridine (Ψ), represent a milestone in combating diseases, thus highlighting Ψ importance in drug development. However, economic and environmental challenges have persisted in sustainable Ψ production. Here, we formulate a streamlined designer Ψ pathway, comprising UMP nucleosidase, ΨMP glycosidase, and ΨMP phosphatase, and realize its gram-scale production by targeted discovery of a prominent UMP-preferred nucleosidase (NmYgdH). The optimized pathway, containing NmYgdH, RjPsuG (ΨMP glycosidase), and HDHD1 (ΨMP-specific phosphatase) is cloned into E. coli and systematic evaluation of multiple strategies achieves a Ψ titer of 44.8 g·L⁻¹. Moreover, a thyA-dependent, tunable, and eco-friendly strategy for sustainable Ψ production is demonstrated in a 5 L bioreactor achieving titer of 45.3 g·L⁻¹. Finally, we establish a simplified-strategy for rapid Ψ purification with a recovery-rate of 71%, and techno-economic analysis is employed to validate the feasibility and advantages of this fermentation platform for Ψ biomanufacturing. Therefore, this study provides a blueprint for industrial-production of nucleoside-related molecules.

Azetidine is a prominent pharmacophore present in dozens of drug-related molecules of both natural and synthetic origins. But how nature builds this moiety has long remained enigmatic. Here we address the full deciphering of a two-metalloenzyme cascade leading to polyoximic acid, an azetidine-containing moiety of the fungicide polyoxin. We demonstrate that the PolE enzyme functions as an Fe²⁺/pterin-dependent l-isoleucine desaturase. Moreover we illustrate that PolF is a new member of the emerging haem-oxygenase-like diiron oxidases, converting the desaturated l-isoleucine to polyoximic acid via an intramolecular C–N cyclization. Remarkably, we also establish that PolF exhibits dual functionality, orchestrating the sequential desaturation and cyclization with l-isoleucine as the initial substrate. Finally, our combined structural and quantum-mechanics/molecular-mechanics studies show that the PolF enzyme employs an extraordinary mechanism for the construction of the azetidine-containing moiety. These findings expand our knowledge on the catalysis of metalloenzymes and open the way for rational access of more azetidine-related molecules.

Type II polyketides (T2PKs) exhibit a wide range of structural diversity and potent pharmacological activities. However, the optimal chassis for the synthesis of T2PKs remains elusive, impeding the effective mining and production of these compounds. In this study, we identify Streptomyces aureofaciens J1-022, a high-yield producer of chlortetracycline, as a promising chassis for T2PKs synthesis. To mitigate precursor competition, we execute an in-frame deletion of two endogenous T2PKs gene clusters, resulting in a pigmented-faded host, designated Chassis2.0. Compared to conventional Streptomyces chassis, Chassis2.0 demonstrates enhanced efficiency in the production of oxytetracycline, achieving a 370% increase relative to commercial production strains. Additionally, the tri-ring type T2PKs, which includes actinorhodin and flavokermesic acid, are synthesized in Chassis2.0 with high efficiency. Furthermore, an unidentified biosynthetic gene cluster (BGC) associated with pentangular T2PKs is directly activated, leading to the production of a structurally distinct TLN-1. In conclusion, we successfully achieve the efficient synthesis of tri-ring type pigmented products, the overproduction of tetra-ring antibiotics, and the discovery of penta-ring type polyketides in Chassis2.0. These findings underscore the potential of Chassis2.0 as an optimal platform for the discovery and overproduction of T2PKs.