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AbstractThe animal gut microbiome is a complex system of diverse, predominantly anaerobic microbiota with secondary metabolite potential. These metabolites likely play roles in shaping microbial community membership and influencing animal host health. As such, novel secondary metabolites from gut microbes hold significant biotechnological and therapeutic interest. Despite their potential, gut microbes are largely untapped for secondary metabolites, with gut fungi and obligate anaerobes being particularly under-explored. To advance understanding of these metabolites, culture-based and (meta)genome-based approaches are essential. Culture-based approaches enable isolation, cultivation, and direct study of gut microbes, and (meta)genome-based approaches utilizeinsilicotools to mine biosynthetic gene clusters (BGCs) from microbes that have not yet been successfully cultured. In this mini-review, we highlight recent innovations in this area, including anaerobic biofoundries like ExFAB, the NSF BioFoundry for Extreme & Exceptional Fungi, Archaea, and Bacteria. These facilities enable high-throughput workflows to study oxygen-sensitive microbes and biosynthetic machinery. Such recent advances promise to improve our understanding of the gut microbiome and its secondary metabolism. Key points• Gut microbial secondary metabolites have therapeutic and biotechnological potential• Culture- and (meta)genome-based workflows drive gut anaerobe metabolite discovery• Anaerobic biofoundries enable high-throughput workflows for metabolite discovery Graphical abstract 

Abstract Designing CRISPR single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we present ALLEGRO, a combinatorial optimization algorithm able to design minimal, yet highly effective sgRNA libraries targeting thousands of species. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting several genes of interest for over 2,000 species across the fungal kingdom. We experimentally validated the sgRNAs designed by ALLEGRO inKluyveromyces marxianus, Komagataella phaffii, andYarrowia lipolytica. In addition, we adopted a generalized Cas9-Ribonucleoprotein delivery system coupled with protoplast transformation to extend ALLEGRO’s sgRNA libraries to other untested fungal genomes, such asRhodotorula araucariae. Our experimental results, along with cross-validation, show that ALLEGRO enables efficient CRISPR genome editing, supporting the development of universal sgRNA libraries applicable to entire taxonomic groups. 

Abstract Drylands comprise 45% of Earth’s land area and contain ecologically critical soil surface communities known as biocrusts. Biocrusts are composed extremotolerant organisms including cyanobacteria, microfungi, algae, lichen, and bryophytes. Fungi in biocrusts help aggregate these communities and may form symbiotic relationships with nearby plants. Climate change threatens biocrusts, particularly moss biocrusts, but its effects on the biocrust mycobiome remain unknown. Here, we performed a culture-dependent and metabarcoding survey of the moss biocrust mycobiome across an aridity gradient to determine whether local climate influences fungal community composition. As the local aridity index increased, fungal communities exhibited greater homogeneity in beta diversity. At arid and hyper-arid sites, communities shifted toward more extremotolerant taxa. We identified a significant proportion of fungal reads and cultures from biocrusts that could not be classified.Rhodotorula mucilaginosaandR. paludigenawere significantly enriched following surface sterilization of healthy biocrust mosses. This aligns with their known roles as plant endophytes. We also observed septate endophyte colonization in the photosynthetic tissues of mosses from arid climates. Collectively, these results suggest that the biocrust mycobiome will undergo significant shifts in diversity due to climate change, favoring extremotolerant taxa as climate conditions intensify. The survey results also highlight taxa with the potential to serve as bioinoculants for enhancing biocrust resilience to climate change. These findings offer valuable insights into the potential impacts of climate change on drylands and provide crucial information for biocrust conservation. 

Abstract Anaerobes thrive in the absence of oxygen and are an untapped reservoir of biotechnological potential. Therefore, bioprospecting efforts focused on anaerobic microbial diversity could rapidly uncover new enzymes, pathways, and chassis organisms to drive biotechnology innovation. Despite their potential utility, anaerobic fermenters are viewed as inefficient from a biochemical perspective because their metabolisms produce fewer ATP (~2) per molecule of glucose processed than heterotrophic respirers (~32–38 ATP). While aerobes excel at ATP generation, they are often less efficient than anaerobes at processes that compete with ATP generation for cellular resources. This perspective highlights how anaerobic adaptations are advantageous for synthetic biology and biomanufacturing applications through the engineering of microbial cell factories. We further highlight emerging applications of anaerobic bioprocessing, including the use of anaerobic metabolisms for lignocellulosic bioprocessing, human and environmental health, and value‐added bioproduction.