De novo design of enzymes that cleave amide bonds represents a major challenge. Here we use RFdiffusion methods to de novo design proteins with water-activating zinc-based active sites adjacent to a peptide substrate binding pocket. Of 135 designs tested, 36% catalyse cleavage of their cognate peptide substrates at the targeted scissile bond. The best design accelerates peptide-bond hydrolysis by more than 108-fold relative to the uncatalysed reaction; four substitutions increase this acceleration to more than 1010-fold by improving active-site preorganization. We use this approach to design metalloproteases that selectively cleave the human disease-associated proteins TDP-43, amyloid-β and serum amyloid A, with rate enhancements of up to 109-fold. We further design caged cytokines and receptor antagonists that are selectively activated by the designed proteases, enabling bioorthogonal control of cell state. Thus, de novo design can yield highly active, programmable proteases with applications in therapeutics, biotechnology and bioremediation. RFdiffusion methods are used to de novo design proteins with water-activating zinc-based active sites adjacent to a peptide substrate binding pocket, yielding highly active, programmable proteases with potential applications in therapeutics, biotechnology and bioremediation.

The alternative sigma factor RpoS (σS) governs stress adaptation in many bacterial clades, yet how it coordinates stress responses with metabolic demands remains poorly understood. Thus, by integrating RNA-seq, ChIP-seq, and physiological and molecular assays, we redefined the RpoS regulon in E. coli, expanding it to 667 genes and demonstrating that metabolic genes unexpectedly predominate over stress-response targets. RpoS directly drives a massive metabolic reprogramming event: it activates glycolytic and sugar transport genes while simultaneously repressing the trichloroacetic acid TCA cycle and oxidative phosphorylation. This metabolic pivot is finely tuned by a novel RpoS-CRP-cAMP-Cra tripartite circuit to balance energy efficiency with survival during stationary phase. Furthermore, we provide a mechanistic framework for RpoS-mediated bidirectional regulation rooted in σS-factor competition, where transcriptional efficiency in the absence of RpoS dictates the regulatory mode: highly efficient promoters are repressed by RpoS displacement, whereas weak promoters are activated. This study advances our understanding of transcriptional precision during bacterial adaptation and offers an architectural blueprint for synthetic metabolic engineering.

By mapping ribosome-protected fragments genome-wide, ribosome profiling (Ribo-seq) has uncovered extensive translation beyond conventional coding sequences, revealing non-canonical ORFs (ncORFs) with roles in diverse biological processes. However, robust ncORF identification remains challenging when Ribo-seq datasets exhibit attenuated periodicity. Protocol-induced biases during library construction is an important contributor to this loss, yet most ORF callers do not explicitly model these distortions. Here, we present RiboBA, a bias-aware probabilistic framework for robust ORF identification from Ribo-seq data. RiboBA combines a generative module that jointly infers protocol-induced biases and codon-level ribosome occupancy with a supervised module that identifies translated ORFs and initiation sites from the bias-adjusted profiles. In simulations with known P-site positions, RiboBA improved exact nucleotide- and codon-level P-site assignment relative to a conventional fixed-offset approach. Across simulations and diverse empirical Ribo-seq datasets, together with orthogonal immunopeptidomics evidence, RiboBA accurately recovers protocol-induced parameters and improves ncORF identification, with pronounced advantages for RNase I libraries with attenuated periodicity, and for MNase and nuclease P1 datasets, while maintaining competitive runtimes. In a Drosophila case study, RiboBA identified candidate ncORFs with coding potential, including recurrent upstream translation in ThrRS and Mettl2, linking ncORF translation to threonine-related translational regulation.

Since the emergence of molecular tension sensors, the understanding of mechanical forces has advanced substantially. However, visualizing molecular tension in mammalian tissues has remained challenging owing to the technical constraints of the Förster resonance energy transfer (FRET)-based molecular tension sensors. Here, we develop a molecular tension sensor based on circularly permuted EGFP with an elastic linker, which is inserted into either αActinin or αCatenin and then fused with mCherry at the C-terminus to simultaneously visualize tension and the amount of sensor protein. This single-fluorophore tension indicator enables subtle tension changes to be visualized simply from color tone under superresolution microscopy. We further generate H11 knock-in mice expressing these indicators, revealing a molecular-specific regulation of mechanical load within tissues. Thus, our molecular tension indicators provide a powerful approach for probing the complex and heterogeneous regulation of mechanical forces in vivo mammalian systems. FRET-free molecular tension indicators enable high-resolution visualization of tension in vivo, revealing that the regulation of mechanical forces is unexpectedly complex, molecule-specific and heterogeneous in live mouse organs.

Archaea play essential roles in global biogeochemical cycles, yet their diversity in environmental samples remains understudied. Limited coverage of universal primers towards archaea, their very low abundance relative to bacteria in the studied samples, inefficient cell lysis and the incompleteness of reference databases complicate archaeal 16S rRNA gene sequencing. Furthermore, standardization lacks due to the absence of commercially available mock communities. Here, we developed a mock community composed of mixtures of DNA from six archaeal and four bacterial strains with known genome sizes and 16S rRNA gene copy numbers. Five mock compositions with different archaeal–bacterial abundance ratios were used to evaluate two universal primer pairs and eight archaea-specific methods. The universal primer pairs detected all taxa but consistently overestimated bacterial abundance, indicating to be less suitable for environments where archaea are more dominant. Archaeal specific primer pairs showed variable specificity, with some exhibiting substantial bacterial co-amplification under certain conditions. The Arch349F–A806R primer pair demonstrated the highest archaeal specificity, the least technical variability, the most consistent agreement with expected mock compositions, and minimal artefactual amplification when applied to a complex bacterial mock community. To show its applicability on environmental samples, this method was applied on Boom Clay pore water samples, which indicated a much higher archaeal diversity than previously shown with a standard commercial method. Overall, this study showed the necessity of primer validation through mock communities and identified Arch349F–A806R as a promising primer pair for archaeal 16S rRNA gene sequencing, especially in samples containing methanogenic archaea.

Restoring endangered ecosystems has become a pressing issue as the effects of global warming continue to harm existing communities. During the last decade, several studies have suggested the use of synthetic biology as a tool to protect these biodiverse communities by increasing their functionality. A critical example concerns soil microbiome communities in drylands, where increasing water retention by some of the constituent species could effectively protect the ecosystem from abrupt degradation. However, how to effectively deploy a functional synthetic construct that can scale its impact to the community level remains an open question. Recent experimental research has designed recombinant gene plasmids with the capacity to horizontally transfer across soil microbial communities. Here, we explore the impacts of synthetic horizontal gene transfer in models of ecological consortia. We define a consumer-resource model in which species can share an engineered plasmid that reduces resource loss, effectively coupling multispecies dynamics and gene spreading. By doing so, we identify a wide range of parameters and the optimal gene transfer conditions for which the intervention promotes biodiversity and biomass gains, while regulating gene propagation and the spread of engineered organisms. Our work provides a first step toward understanding the mechanisms and opportunities of engineered plasmid transfer in multispecies ecological communities.

The number of internal repeat modules remains a major limiting factor in the construction and functional exploration of long repeat proteins. As a solution, we present High-Utility Recursive Directional Ligation for Extended Repeat proteins (HURDLER)—a modular cloning strategy combining codon optimization, seamless insertion of restriction sites, and modified recursive directional ligation. It presents a very robust technique to clone DNA for large repeat proteins, which may also contain non-repetitive parts, with high fidelity requiring a minimal number of individual cloning steps. Using HURDLER, we cloned designed armadillo repeat proteins with up to 85 internal modules, corresponding to >3700 residues. The method also generates intermediate variants with defined numbers of internal repeats, enabling systematic investigation of how the number of modules influences protein structure and function. HURDLER thereby provides a reliable solution to overcome restrictions in the overall number of internal modules used in the construction of very long repeat proteins. Such proteins are increasingly utilized as building blocks in macromolecular protein assemblies with defined structures.

Episomal plasmids are widely used in yeast metabolic engineering for their flexibility and high transformation efficiency. However, their application in Yarrowia lipolytica is often limited by low copy number and insufficient genetic stability. In this study, a robust episomal plasmid that balanced genetic stability and protein expression capacity was developed in Y. lipolytica. The autonomously replicating sequence (ARS) elements were selected, and ARS1 was identified as a favorable element that balanced genetic stability and gene expression capacity. To improve plasmid copy number, different ubiquitin degrons were used to tune selection marker abundance, and Ubi-R-Δk-mediated marker resulted in an approximately fourfold increase in plasmid copy number (from ∼1 to ∼5 copies per cell). The copy number of the engineered plasmid was one of the highest‑copy episomal systems in Y. lipolytica. To further evaluate the engineered plasmid, we applied it to β‑carotene biosynthesis. The results showed that the β‑carotene titer was 3.43‑fold higher than that of the non‑optimized plasmid. Collectively, this work expands the episomal plasmid toolbox for Y. lipolytica and establishes a generalizable framework for robust protein expression plasmid engineering in non-conventional yeasts.

Nanopore protein sequencing has advanced from a theoretical ambition to a practical technology, overcoming major challenges in protein unfolding, transport and signal detection to achieve single-amino-acid and post-translational modification sensitivity. With both full-length and peptide-based approaches progressing towards commercialization, the field is now focused on scaling throughput and refining analysis tools to unlock a new generation of single-molecule proteomics.

Curiosity-driven studies of lipid organization and lipid asymmetry in biological membranes led to scalable methods for making liposomes, efficient loading of drugs and ionizable cationic lipids. In turn, these advances led to lipid nanoparticles enabling the RNA medicine Onpattro and the COVID-19 mRNA vaccines, and they are now facilitating gene therapies that can potentially treat most human diseases. lnp