Bradyrhizobium and Sinorhizobium are dominant soybean microsymbionts in acidic/neutral and alkaline soils, respectively. However, the molecular mechanisms underlying this pH-dependent adaptation remain elusive. In this study, phylogenomic analysis of 286 Bradyrhizobium and 322 Sinorhizobium genomes revealed that Bradyrhizobium possesses abundant xeno-siderophore receptors but has limited siderophore biosynthesis functions. In contrast, gene clusters directing siderophore biosynthesis are enriched in Sinorhizobium. As siderophores can chelate the prevalent insoluble Fe3+ under neutral and alkaline conditions, whereas being less important in acidic environments where soluble Fe2+ is readily accessible, we hypothesized that the genus-dependent phyletic distribution of siderophore biosynthesis and exploitation functions may contribute to the pH adaptation of these two genera. Indeed, Bradyrhizobium species barely grow under iron-limiting conditions, and this growth defect can be rescued by xeno-siderophores produced by Sinorhizobium. Using a xeno-siderophore-exploiting Bradyrhizobium diazoefficiens strain, an engineered xeno-siderophore exploiter, and an altruistic siderophore-producing strain derived from Sinorhizobium fredii, we revealed the competitive advantage of xeno-siderophore exploitation during soybean nodulation. Heterologous expression of certain Bradyrhizobium xeno-siderophore receptors, along with various adaptive mutations in the genome of the S. fredii receptor-lacking mutant, allowed this mutant to rapidly restore growth under iron-limiting conditions. These adaptive events in experimental evolution depend on the siderophore biosynthetic function of S. fredii. Taken together, these findings suggest that the siderophore utilization ability of soybean rhizobia can be positively selected under iron-limiting conditions: by maintaining abundant xeno-siderophore receptors in acid-tolerant Bradyrhizobium and by the rapid adaptive evolution of utilization machinery for self-produced siderophores in alkaline-tolerant Sinorhizobium.

Class 1 CRISPR–Cas systems utilize multi-subunit effector ribonucleoprotein complexes to identify and target DNA. Upon recognition, type I systems recruit the helicase/nuclease Cas3 for DNA degradation, while type IV-A systems use the helicase CasDinG for transcriptional repression. Here, we developed two recombinant class 1 CRISPR–Cas genome editing tools for inducing large genomic deletions: the compact type I-Fv (also termed I-F2) system from Shewanella putrefaciens and the type IV-A1 system from Pseudomonas oleovorans. In the latter, CasDinG was engineered to include a C-terminal HNH nuclease domain, conferring DNA cleavage activity and enabling analysis of CasDinG processivity. Whole-genome sequencing of Escherichia coli BL21-AI was used to monitor genome reduction and DNA repair mechanisms in response to CRISPR–Cas-induced damage. Small deletions were flanked by microhomologies, consistent with repair via alternative end joining, whereas deletions larger than 10 kb consistently terminated at nearby IS1 elements, implicating these sequences in the repair process. This study introduces compact type I and engineered type IV-A genome editing tools with distinct protospacer-adjacent motif requirements and provides new insights into CasDinG evolution and the DNA repair pathways engaged during CRISPR–Cas-mediated genome editing.

The Gene Ontology (GO) knowledgebase https://geneontology.org is a comprehensive resource describing the functions of genes. The GO knowledgebase is regularly updated and improved. We describe here the major updates that have been made in the past 3 years. The ontology and annotations have been expanded and revised, particularly in several areas of biology: cellular metabolism, multi-organism interactions (e.g. host-pathogen), extracellular matrix proteins, chromatin remodeling (e.g. the “histone code”), and noncoding RNA functions. We have released version 2 of a comprehensive set of integrated, reviewed annotations for human genes, which we call the “functionome.” We have also dramatically increased the number of GO-CAM models, with over 1500 models of metabolic and signaling pathways, primarily in human, mouse, budding and fission yeast, and fruit fly. Finally, we discuss our current recommendations and future prospects of AI in the use and development of GO.

Colouration of textiles is responsible for 3% of global greenhouse gas emissions and 20% of global wastewater generation. An estimated 20% of the dyes and pigments used in textile colouration is lost to wastewater. Synthetic dyes and pigments, along with many of the chemicals added to their formulations, are

nonbiodegradable, persistent, and bioaccumulative in aquatic

ecosystems, posing signi cant risks throughout the food chain. Regulatory bodies in both Europe and the United States are continuing to ban, restrict, and phase out many synthetic dyes and pigments. The EU recently banned a significant number of common textile dyes known to release carcinogenic compounds,

while in the United States, 8 common synthetic food dyes are

being targeted for immediate ban in the US food supply, with the FDA planning to phase out all remaining synthetic food dyes. While bio-based alternatives are available on the market, they are

currently more expensive, restricting their application to niche product applications that can absorb the price premium. The total market size for dyes and pigments is more than US$ 50 billion and is growing at a rate of 3–10% annually. The ‘cost of being unsustainable’ is increasing rapidly, particularly in the EU, as new regulations make the use of hazardous chemicals more difficult

and expensive. This is driving a shift toward the use of more sustainable chemicals.

Cyclic disulfide-rich peptides have become increasingly popular in drug development because their structures enhance molecular stability and allow for mutagenesis to introduce non-native functions. This review focuses on yeast-based platform technologies and their utility in advancing cyclic disulfide-rich peptides as drug modalities and for large-scale biomanufacturing. These technologies include yeast surface display which facilitates the screening of large libraries to develop peptide binders with strong affinity and selectivity for protein targets, while maintaining the innate high stability of the peptide scaffold via protease-based selection pressure. We also describe a recently developed platform that leverages yeast’s ability to secrete correctly folded disulfide-rich peptides while simultaneously displaying peptide or protein tags on their surfaces. In combination with microfluidics technology, the platform creates single-cell yeast-in-droplets reactors, enabling the screening of large libraries based on functional output rather than solely on binding affinity. After identifying cyclic peptide candidates through library-based discovery, these candidates can be produced using a versatile yeast-based bioproduction platform. Traditionally, cyclic disulfide-rich peptides are produced through solid-phase synthesis, a method that generates significant amounts of toxic waste. In contrast, yeast-based bioproduction offers an environmentally sustainable alternative. It has the capability to produce structurally distinct peptides with minimal adjustments and is easily scalable using microbial fermenters, making it an ideal choice for large-scale production.

Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the correct edits, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known “inhibitor” Acr protein can unexpectedly acting as an “enhancer” of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity. Prime editing enables precise genetic modification but often suffers from unwanted byproducts. Here, authors show that the anti-CRISPR protein AcrIIA5 unexpectedly enhances prime editing efficiency while reducing unintended indels, offering an effective strategy to improve activity and specificity.

Directed evolution facilitates functional adaptations through stepwise changes in sequence that alter protein structure. While most campaigns yield solutions that maintain the framework of a rigid protein architecture, a few have produced enzymes with more notable structural differences. One example is a polymerase that was evolved to synthesize threose nucleic acid (TNA) with near-natural activity. Understanding how this enzyme arose provides a model for studying pathways that guide enzymes toward more productive regions of the fitness landscape. Here, we trace the evolutionary trajectory of an unnatural polymerase by solving crystal structures of key intermediates along the pathway and evaluating their biochemical activity. Contrary to the view that fidelity is a product of increased catalytic efficiency, we find that accuracy and catalysis are decoupled activities guided by separate ground-state and transition-state discrimination events. Together, these results offer a glimpse into the forces responsible for shaping the emergence of new enzyme functions. Engineering polymerases to synthesize alternative genetic polymers remains a challenging problem in synthetic biology. The current study offers insights into the structural and biochemical changes responsible for improving the fidelity and catalytic activity of a laboratory evolved TNA polymerase.