Phages are important drivers of bacterial evolution and have potential as antimicrobials. However, incomplete understanding of phage biology and our inability to rapidly engineer them with new genetic cargo hinder progress towards phage-based therapies. Here we develop an unbiased, genome-wide mutational tool for phages. This approach, phage Tn-seq, uses Tn5 transposon mutagenesis using anti-CRISPR-based selection and deep-sequencing. Phage Tn-seq was successful across diverse phages, including a nucleus-forming jumbo phage and enabled gene essentiality assignment consistent with phage structural proteomics and core gene conservation. In addition, insertion biases allowed prediction of transcriptional direction and early injected and highly expressed regions. We exploit transposons to deliver new cargo to phages within a few days and created an orthogonal artificial intelligence-designed Acr transposon system to generate phage double mutants. Transposon insertion was also achieved in phages with hypermodified DNA. Phage Tn-seq is a versatile tool to advance our understanding and application of phages. Phage Tn-seq uses Tn5 transposon mutagenesis with anti-CRISPR-based selection and deep sequencing as a method to generate unbiased, genome-wide mutations across diverse phages

Diverse chemicals biosynthesis demand distinct precursor acetyl-CoA and energy inputs, yet strategies balancing carbon atom economy and energy generation for varied products remain scarce. Here, we design an autonomous hybrid energy-carbon balancing apparatus (ATHENA) to establish a paradigm for redox-driven, dynamically adaptive regulation of carbon conservation and energy synthesis in E. coli. ATHENA features modularization of acetyl-CoA and NADH synthesis pathways for carbon conservation and energy generation, paired with NADH-responsive positive and negative genetic circuits constructed using the BsRex sensor and antisense RNA. This system enables intracellular redox state-dependent rational metabolic flux allocation to balance carbon atom economy and energy generation. Ultimately, ATHENA significantly improves yields and titers of five products with differential acetyl-CoA and energy demands. In the 5-L bioreactor, acetate yield markedly exceeds its native theoretical yield, while that of mevalonate nearly approaches this theoretical upper limit. This work provides a universal strategy for constructing high carbon-yield microbial chassis cells. Metabolic engineering strategies that balance carbon atom economy and energy generation remain scarce. Here the authors engineer E. coli with an NADH-responsive genetic circuit to regulate carbon-conserving and energy-generating modules, improving yields and titres of diverse products.

Nitrification is a key process in the global nitrogen cycle, yet the physiological characterization of nitrifiers remains limited due to their low culturability. Although culture-independent approaches have provided important ecological insights, they do not fully resolve the physiological properties of nitrifying microorganisms. Here, we show that common gelling agents (agar, agarose, and gellan gum) contain toxic substances that inhibit the growth of diverse nitrifying microorganisms as well as some heterotrophic bacteria. These inhibitory substances appear to be generated during autoclaving and act via mechanisms distinct from previously reported hydrogen peroxide. Removal of these inhibitory factors using a simple washing strategy enables colony formation in recalcitrant nitrifiers, including Nitrososphaera (ammonia-oxidizing archaea) and Nitrospira (nitrite-oxidizing bacteria). Application of this approach to environmental samples enabled the isolation of multiple difficult-to-culture nitrifying strains, including ammonia-oxidizing and nitrite-oxidizing microorganisms. Together, these findings demonstrate that the limited culturability of nitrifiers on solid medium is largely driven by gelling agent–derived inhibitory substances and can be overcome by a simple and broadly applicable strategy.

Across cultivation, processing, and consumption, tea quality and bioactivity are shaped by plant-microbiota interactions. This review develops a field-to-gut framework centered on the tea plant holobiont and downstream microbial partners. Within that frame, rhizosphere and endophytic microbiota are examined as regulators of flavor precursors, host metabolism, and defense. Processing microbiota are considered for remodeling tea chemistry and gut microbiota for bioactivating tea compounds, with microbial transmission, ecological replacement, and metabolite carryover distinguished and translational directions identified in microbiome-guided cultivation, controlled fermentation, and metabotype-informed nutrition.

FAIDR-Desi is a non-technical user interface for design of intrinsically disordered regions (IDRs) based on greedy optimization of sequence features (simple sequence statistics correlated with IDR function, including amino acid composition, residue patterning and short linear interaction motifs), using more features (127) than other IDR design approaches. FAIDR-Desi can be used to design synthetic IDR mimics to establish feature sufficiency for function, or to knock out features to show necessity for function, exemplified with experiments on the yeast Cox15 mitochondrial targeting signal.

Protein synthesis forms the very basis of life. Cells rely on a sophisticated machinery of factors for the translation of an RNA template into a correct amino acid sequence. We owe a lot of our understanding of this process to specific small-molecule modulators. Since the discovery of antibiotics inhibiting bacterial protein synthesis, small molecules have served as probes to dissect the translation apparatus and as medications to fight disease. Advances in sequencing-based techniques and structural methods now provide for a highly detailed mechanistic understanding. Recent evidence shows that many translation modulators act in an RNA- or a peptide-sequence-selective manner. These molecules present exciting opportunities to better understand the translation machinery and provide new venues to modulate the expression of certain protein subpopulations. In this Review, we summarize recent findings on sequence-selective translation modulators, with emphasis on eukaryotes, the techniques that enabled their discovery, and offer an outlook into their potential applications. Small-molecule modulators of protein synthesis are invaluable tools for dissecting the translation apparatus. This Review summarizes the mechanisms of molecules that disrupt translation in an amino acid or mRNA sequence-dependent manner, with focus on eukaryotic systems. It discusses relevant techniques used in their characterization and offers perspectives into their potential applications.

Overlapping genes are widely distributed across the genomes of nearly all living organisms. Although thousands of overlapping genes have been identified and studied in several species, a comprehensive annotation of these genes is lacking, and the forces shaping their evolution remain poorly understood. Here, we present a comparative analysis of overlapping genes in 253 prokaryotic genomes, 115 plant genomes, and 111 animal genomes. We find that co-oriented overlapping genes predominate in prokaryotes, whereas nested overlapping genes are the most common type in eukaryotes. In prokaryotes, overlapping genes are significantly shorter than non-overlapping genes, and 69% of co-oriented overlaps occur in translational phase 2. The number of overlapping genes is strongly correlated with genome size in prokaryotes but not in eukaryotes. We estimated the rates at which overlapping genes are gained and lost over evolutionary time and found that loss of overlapping orthologs is the highest rate in eukaryotes, whereas the highest rate in prokaryotes is the gain of overlapping genes from adjacent genes. Expression analyses revealed that, compared with neighboring genes, nested genes exhibit lower expression levels, altered expression correlations, and higher tissue-specific expression patterns in selected eukaryotic species. Based on these patterns, we propose distinct models to explain the evolutionary pressures acting on overlapping genes in prokaryotes and eukaryotes. Together, our findings provide new insights into the evolution of overlapping genes.

Microbial CO₂ assimilation provides a strategy for sustainable biomanufacturing. However, CO₂ reduction requires substantial energy input, while native energy systems are tightly regulated and extensively consumed by endogenous metabolism, limiting assimilation efficiency. Here, we design and construct an orthogonal energy system in E. coli, achieving an intracellular NUDH concentration exceeding 7.0 mM and an NUDH/NUD⁺ ratio above 55. Subsequently, we design a CO₂-formate-acetyl-CoA-pyruvate-malate pathway and reprogram key enzymes for NUDH-dependent operation. Integration of the OES-driven CAM* pathway with a rationally rewired native electron transport chain supports growth of the engineered E. coli on CO₂ and methanol, with a doubling time of 8.6 ± 0.3 h and a maximum OD₆₀₀ of 37.5 ± 5.8. Moreover, the platform extends CO₂ assimilation to biosynthesis of multiple chemicals from distinct CAM* pathway nodes, including mevalonate, lactate, 2,3-butanediol, and succinate. Together, these results establish a framework for reprogramming microbial energy metabolism and expanding one-carbon biotransformation. Microbial CO₂ reduction requires substantial energy input which limits assimilation efficiency. Here the authors create a dedicated reducing-power supply for CO₂ assimilation in E. coli and couple this with methanol oxidation to support the production of four chemicals from distinct metabolic nodes.

Maintaining microbially mediated phosphorus mobilization under intensive nitrogen fertilization is difficult in acidified paddy soils. Using a decade-long field experiment in a rice–oilseed rape rotation, we compared an ordered manure-substitution series, in which manure input increased while mineral nitrogen input decreased, with a mineral nitrogen dose gradient. Increasing mineral nitrogen input was associated with lower diversity and lower absolute abundance of the phoD-harboring bacterial guild and a marked shift in community composition. Across the manure-substitution series, soil pH increased from 5.94 to 6.62, potential alkaline phosphatase activity increased by up to 158%, and Olsen-extractable phosphorus increased by up to 182%. The diversity, composition, and absolute abundance of the phoD guild changed progressively across the same series. Distance-based redundancy analysis identified soil pH as the strongest measured marginal correlate of community composition, whereas variation partitioning indicated that the effects of pH, carbon, and phosphorus were largely shared. An exploratory structural equation model was consistent with conditional associations of both community composition and absolute gene abundance with alkaline phosphatase activity, which was, in turn, associated with Olsen-extractable phosphorus. The highest observed means for gene abundance, grain yield, and grain phosphorus removal occurred in the intermediate manure treatment, whereas treatment-level copper and zinc concentrations increased towards the highest-manure treatment. Together, these findings indicate that manure substitution can counteract mineral nitrogen-associated deterioration of the phoD guild and support microbial phosphorus mobilization, although the agronomic and metal responses require confirmation across sites and seasons.

Skeletal muscle has a profound influence on metabolic health, functional capacity and resilience across the lifespan. Beyond dietary protein and physical activity, the gut microbiome may have the capacity to impact skeletal muscle mass and function through the bidirectional network termed the gut-muscle axis (GMA). Fermented protein foods (FPFs) are protein rich matrices transformed by microbial activity that integrate modified protein structures, bioactive peptides, and live microorganisms, or components thereof, which could modulate skeletal muscle physiology. Here, we review mechanistic, preclinical and human evidence concerning the potential of FPFs to influence skeletal muscle health through GMA modulation. Although emerging human studies suggest favourable effects on metabolic regulation, inflammatory pathways and gut microbial ecology, direct evidence that FPFs impact muscle protein synthesis, muscle mass or function remains limited. Furthermore, the literature is characterised by considerable heterogeneity in interventions and outcome measures. Studies integrating comprehensive microbiome characterisation, multi-omics approaches and direct skeletal muscle phenotyping are needed to determine whether FPFs confer advantages over conventional protein foods or more established microbiome targeted strategies such as probiotic supplementation. Such studies will be essential to elucidate their role in targeted nutritional strategies for ageing, metabolic disease, muscle atrophy, and physical performance.