Despite advances in mass spectrometry and emerging single-molecule approaches, sequencing peptides at the single-molecule level remains a central challenge in proteomics. Here we present a ‘reverse translation’ strategy that enables single-molecule peptide sequencing with single-amino-acid resolution. In this approach, peptides undergo a modified Edman degradation that iteratively releases N-terminal amino acids tagged with peptide-specific DNA barcodes. Antibody-mediated proximity extension assays identify these barcoded amino acids and generate PCR-amplifiable DNA reporters that record the identity, position and originating peptide of each amino acid. The resulting DNA library is directly read by high-throughput sequencing, converting peptide sequences into digital DNA outputs. Using this approach, we demonstrate true single-molecule peptide sequencing, achieving full sequence coverage in millions of reads and accurate differentiation of both native and post-translationally modified peptides. These results establish a framework that redefines protein sequencing as a DNA sequencing problem and lays the foundation for high-throughput, de novo single-molecule protein sequencing. Peptides are sequenced by converting each amino acid into amplifiable DNA barcodes.

The mushroom innovation ecosystem highlights significant advances in mycology, biotechnology, and biofabrication, as well as the role of startups in translating these breakthroughs into products. By tracing these translational pathways, this Perspective aims to demonstrate that mushrooms represent more than a scientific resource - they embody a cross-sectoral model for bioinspired innovation with profound impact on sustainable industry and human wellbeing. This Review article explores how startups translate advances in mycology, biotechnology and biofabrication to address pressing environmental and societal challenges. It positions mushrooms as a cross-sector platform for sustainable innovation with wide-reaching benefits for industry and human wellbeing.

Tumor immunotherapy is often compromised by an immunosuppressive tumor microenvironment (TME) characterized by abnormal vasculature and exhausted T cells. Here, given the role of nitric oxide (NO) in favorably remodeling the TME, we engineered Escherichia coli Nissle 1917 (ECN) with a synthetic arginine–NO circuit (ECN-NO) that modifies the arginine synthesis pathway to constitutively synthesize arginine and enable sustained NO production. Specifically, deletion of the arginine repressor ArgR relieved feedback inhibition of arginine biosynthesis, whereas co-expression of argininosuccinate synthase and lyase (ArgG/ArgH), together with Bacillus subtilis nitric oxide synthase (BsNOS), enabled sustained NO production through enhanced arginine regeneration. Intratumoral colonization of ECN-NO significantly enhanced the antitumor efficacy of anti-programmed cell death ligand 1 (αPD-L1) immunotherapy, resulting in durable tumor regression across multiple solid tumor mouse models. Mechanistically, ECN-NO induced vascular normalization and dendritic cell recruitment, alleviated tumor immunosuppression and synergized with αPD-L1 to expand functional CD8+ T cells, reverse T cell exhaustion and promote memory T cell formation, establishing antitumor immunity for at least 120 days. Solid tumors are sensitized to anti‑PD‑L1 immunotherapy by engineered E. coli to produce nitric oxide.

Alternative splicing (AS) has emerged as a regulatory layer in plant adaptation to the environment. In particular, biotic stresses trigger a drastic remodeling of the plant AS landscape, with minimal overlap with changes at the gene expression level, suggesting an additional, albeit poorly understood, mechanism of regulation. Recent studies have revealed that effectors from unrelated pathogens target core spliceosome components as well as accessory splicing factors. While this targeting is beginning to shed light on the relevance of the modulation of the plant AS landscape for pathogen invasion, it has also led to the identification of novel splicing factors, allowing the discovery of unexplored characteristics of the plant splicing machinery. Here, we review this emerging field, which delineates an additional battleground in the evolutionary arms race between plants and pathogens and has the potential to advance our biochemical and mechanistic understanding of the plant spliceosomal complex.

Chemical innovation is essential for fungi to adapt to ever-changing ecological environments. However, the environmental and evolutionary drivers of fungal metabolic differentiation remain ambiguous. Here, we show the phylogeographic diversity of 1052 Aspergillus flavus strains across four continents, as conducted through phylogenetic and biogeographical analysis, including 544 newly sequenced strains from China. These strains exhibit varying levels of population-specific mycotoxin production, as determined by population metabolomics analysis. We report a toxigenic subpopulation from China, identified through comparative population genomics analysis. Pan-metabolome analysis reveals strong phylogeographic metabolic patterns associated with specific ecological niches. Low-mycotoxin production clades harbor distinct uncharacterized biosynthetic gene clusters and produce different specialized metabolites instead. This discrepancy is only partially explained by variation in biosynthetic pathway genes, and changes in regulation and primary metabolism appear to mainly drive differentiation of specialized metabolite profiles across fungal populations, as indicated by pangenome profiling, metabolites-genome-wide association study, genotype-environment association study, pan-transcriptome analysis, and gene knockout experiments. Altogether, our results reveal how environmental shifts drive the fungal metabolic evolution, and provide insights for predicting the risk of harmful fungal outbreaks and for biogeographically-informed, precise control measures. Drivers of fungal metabolic diversity are incompletely understood. Here, the authors conduct a global genomics study of over 1,000 pathogenic fungi to show that geography shapes the metabolic diversity in Aspergillus flavus revealing how climate drives fungal chemical adaptive evolution.

Diverse phage-bacteria communities coexist at high densities in environmental, agricultural, and human-associated microbiomes. Phage-bacteria coexistence is often attributed to coevolutionary processes mediated by complex, pairwise infection networks. Here, using in vitro experiments and mathematical models, we explore how higher-order interactions function as a complementary, ecological feedback mechanism to stabilize phage-bacteria communities. To do so, we examine an environmentally-derived, synthetic phage-bacteria community comprised of five marine heterotrophic bacteria (Cellulophaga baltica and Pseudoalteromonas strains) and five associated phage. We used Bayesian inference to reconstruct free phage production in one-step growth experiments and then forecasted pairwise phage-bacteria community dynamics over multiple infection cycles. In contrast to model predictions of rapid bacterial population collapse, each bacterial strain persisted in the community. We hypothesized and then experimentally validated the relevance of infection attenuation at relatively high viral densities. We extended models into a community context, corroborating complex coexistence of all phage and bacteria. Life history traits inferred in community fits often differed from those inferred in a pairwise context, implicating higher-order interactions as an additional, ecological stabilization mechanism. Follow-up experiments confirm that phage traits (including burst size) can shift when infecting single vs. multiple strains. More broadly, these findings suggest that complex community coexistence of phage and bacteria may be more common than anticipated when including feedback mechanisms outside of the growth-dominated regimes of fitted pairwise models that do not reflect the full scope of ecologically relevant contexts.

Recent advances in genetic engineering have provided diverse tools for artificially diversifying both prokaryotic and eukaryotic cell populations1–6. However, achieving precise control over the ratios of multiple cell types within a population derived from a single founder remains a major challenge. Here we introduce a suite of recombinase-mediated genetic devices designed to accurately control population ratios, enabling the distribution of distinct functionalities across multiple cell types. We systematically evaluated key parameters that influence recombination efficiency and developed data-driven models to reliably predict binary differentiation outcomes. Using these devices, we constructed parallel and series circuit topologies to implement user-defined, multistep cell-fate branching programs. The branching devices facilitated the autonomous differentiation of precision fermentation consortia from a single founder yeast strain, optimizing cell-type ratios for applications such as pigmentation and cellulose degradation. Similar effects were obtained with mammalian cells. We also engineered multicellular aggregates with genetically encoded morphologies by coordinating self-organization through cell adhesion molecules. Our work provides a comprehensive characterization of recombinase-based cell-fate branching mechanisms and introduces an approach for constructing synthetic consortia and multicellular assemblies. A synthetic genetic circuit made up of recombinase-based cell-fate branching devices enables precise control over the ratios of cell types in an offspring population derived from one founder strain, and could be used to build user-defined multicellular aggregates.

Photosensory protein domains, derived from nature, are foundational for optogenetic protein engineering. Tailoring their properties enables their full exploitation for optogenetic regulation in basic research and applied bioengineering applications. Here, we present a simple, yet powerful strategy based on random mutagenesis coupled to high-throughput screening that allowed altering the most fundamental properties of the widely used nMag/pMag photodimerization system: its light sensitivity and activation. Variants were characterized in vivo in bacteria by flow cytometry and during the entire growth curve by spectrofluorometry. We identify mutations that either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the light activation and dark-to-light fold change. Notably, light sensitivity and activation levels could be changed independently. In addition, we demonstrated that the shapes of the dose-response curves can be finely tuned. This broadens the applicability of the Magnets photosensors for optogenetic regulation strategies. Photosensory protein domains, derived from nature, are foundational for optogenetic protein engineering. Here the authors develop a high-throughput strategy to tune the light sensitivity and activation of a widely used optogenetic protein system.

Subcellular compartments organize RNAs into phase-separated condensates, significantly influencing RNA metabolism. However, the study of how specific RNAs regulate interacting factors and their phenotypic outcomes is hindered by the lack of advanced imaging and regulation tools. To address this, we developed an orthogonal RNA aptamer system, Clivia-HT, which integrates a fluorescent imager with a ribonuclease-targeting chimera (RIBOTAC) degrader. This platform enables simultaneous RNA visualization and targeted degradation in living cells. Furthermore, we engineered light-activatable and light-inactivatable RIBOTACs to achieve temporal RNA degradation control. Applying this system, we demonstrated that Activating Transcription Factor 4 (ATF4) mRNA localizes to stress granules and regulates their dynamics. We also detected the role of Non-Coding RNA Activated by DNA Damage (NORAD) in RNA–protein condensate assembly. Our approach establishes a versatile method for spatiotemporal RNA manipulation, providing a powerful tool for probing RNA function in dynamic cellular environments. Studying RNA in cellular condensates requires precise tools. Here, the authors developed an orthogonal RNA aptamer system for simultaneous RNA visualization and targeted degradation, revealing new roles for specific RNAs in stress granule and NP body dynamics.