The genomes of filamentous fungi contain up to ~90 biosynthetic gene clusters (BGCs), encoding diverse secondary metabolites, an enormous reservoir of untapped chemical potential. However, recalcitrant genetics, cryptic expression, and unculturability prevent the systematic exploitation of these gene clusters and harvesting of their products. With heterologous expression of fungal BGCs largely limited to expression of single or partial clusters, we established a scalable process for expression of large numbers of full-length gene clusters, called FAC-MS. Using Fungal Artificial Chromosomes (FACs) with Metabolomic Scoring (MS) we screened 56 secondary metabolite BGCs from diverse fungal species for expression in A. nidulans. Fifteen new metabolites were discovered and confidently assigned to their BGCs. A new macrolactone, valactamide A, and its hybrid PKS-NRPS gene cluster were characterized extensively using this integrated platform. Regularizing access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Fungi are prolific producers of natural products, compounds which have had a large societal impact as pharmaceuticals, mycotoxins, and agrochemicals. Despite the availability of over 1,000 fungal genomes and several decades of compound discovery efforts from fungi, the biosynthetic gene clusters (BGCs) encoded by these genomes and the associated chemical space have yet to be analyzed systematically. Here, we provide detailed annotation and analyses of fungal biosynthetic and chemical space to enable genome mining and discovery of fungal natural products. Using 1,037 genomes from species across the fungal kingdom (e.g., Ascomycota, Basidiomycota, and non-Dikarya taxa), 36,399 predicted BGCs were organized into a network of 12,067 gene cluster families (GCFs). Anchoring these GCFs with reference BGCs enabled automated annotation of 2,026 BGCs with predicted metabolite scaffolds. We performed parallel analyses of the chemical repertoire of fungi, organizing 15,213 fungal compounds into 2,945 molecular families (MFs). The taxonomic landscape of fungal GCFs is largely species specific, though select families such as the equisetin GCF are present across vast phylogenetic distances with parallel diversifications in the GCF and MF. We compare these fungal datasets with a set of 5,453 bacterial genomes and their BGCs and 9,382 bacterial compounds, revealing dramatic differences between bacterial and fungal biosynthetic logic and chemical space. These genomics and cheminformatics analyses reveal the large extent to which fungal and bacterial sources represent distinct compound reservoirs. With a >10-fold increase in the number of interpreted strains and annotated BGCs, this work better regularizes the biosynthetic potential of fungi for rational compound discovery.

Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.

Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospecive highlights various molecularfamilies with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products.