Microorganisms produce a wide range of natural products (NPs) with clinically and agriculturally relevant biological activities. In bacteria and fungi, genes encoding successive steps in a biosynthetic pathway tend to be clustered on the chromosome as biosynthetic gene clusters (BGCs). Historically, “activity-guided” approaches to NP discovery have focused on bioactivity screening of NPs produced by culturable microbes. In contrast, recent “genome mining” approaches first identify candidate BGCs, express these biosynthetic genes using synthetic biology methods, and finally test for the production of NPs. Fungal genome mining efforts and the exploration of novel sequence and NP space are limited, however, by the lack of a comprehensive catalog of BGCs encoding experimentally-validated products. In this study, we generated a comprehensive reference set of fungal NPs whose biosynthetic gene clusters are described in the published literature. To generate this dataset, we first identified NCBI records that included both a peer-reviewed article and an associated nucleotide record. We filtered these records by text and homology criteria to identify putative NP-related articles and BGCs. Next, we manually curated the resulting articles, chemical structures, and protein sequences. The resulting catalog contains 197 unique NP compounds covering several major classes of fungal NPs, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids. The distribution of articles published per compound shows a bias towards the study of certain popular compounds, such as the aflatoxins. Phylogenetic analysis of biosynthetic genes suggests that much chemical and enzymatic diversity remains to be discovered in fungi. Our catalog was incorporated into the recently launched Minimum Information about Biosynthetic Gene cluster (MIBiG) repository to create the largest known set of fungal BGCs and associated NPs, a resource that we anticipate will guide future genome mining and synthetic biology efforts toward discovering novel fungal enzymes and metabolites.

Pyrazomycin is a rare C‐nucleoside antibiotic containing a naturally occurring pyrazole ring, the biosynthetic origin of which has remained obscure for decades. In this study we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomyces candidus NRRL 3601, revealing that the StrR‐family regulator PyrR is the cluster‐situated transcriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stable‐isotope feeding experiments provide support for the hypothesis that PyrN is a new nitrogen–nitrogen bond‐forming enzyme that catalyzes the linkage of the ϵ‐NH2 nitrogen atom of l‐N6‐OH‐lysine and the α‐NH2 nitrogen atom of l‐glutamic acid. This study lays the foundation for further genetic and biochemical characterization of pyrazomycin pathway enzymes involved in constructing the characteristic pyrazole ring.

Pyridoxal 5′‐phosphate (PLP) is an organic cofactor employed by ~ 4% of enzymes. The structure of the PLP cofactor allows for the stabilization of carbanions through resonance. A small number of PLP‐dependent enzymes employ molecular oxygen as a cosubstrate. Here, we review the biological roles and possible mechanisms of these enzymes, and we observe that these enzymes are found in multiple protein families, suggesting that reaction with oxygen might have emerged de novo in several protein families and thus could be directed to emerge again through laboratory evolution experiments.

Pyrazomycin is ar are C-nucleoside antibiotic containing an aturally occurring pyrazole ring, the biosynthetic origin of which has remained obscure for decades. In this study we report the identification of the gene cluster responsible for pyrazomycin biosynthesis in Streptomycesc andidus NRRL 3601, revealing that the StrR-family regulator PyrR is the cluster-situatedt ranscriptional activator governing pyrazomycin biosynthesis. Furthermore, our results from in vivo reconstitution and stableisotope feeding experiments provides upport for the hypothesis that PyrN is an ew nitrogen-nitrogenb ond-forming enzyme that catalyzes the linkageo ft he e-NH 2 nitrogen atom of l-N 6 -OH-lysine and the a-NH 2 nitrogen atom of l-glutamic acid. This study lays the foundation for further genetic and biochemicalc haracterization of pyrazomycin pathway enzymes involved in constructing the characteristic pyrazolering.Natural products containing nitrogen-nitrogen (NÀN) bonds are ag roup of specialized metabolites with diverses tructures and av ariety of biological activities. [1] These compounds have been isolated from differents ources, including bacteria, fungi, and plants.Despitee xtensive studies on the genetic and biochemical basis of naturalp roduct biosynthesis over the past three decades, the biochemical routes leading to enzymatic NÀNb ond formation are only startingt ob er evealed. [2][3][4][5][6][7][8][9][10][11][12][13][14] We recently reported ah eme-dependent piperazate synthase responsible for NÀNc yclization to give piperazic acid, ab uilding block for many nonribosomal peptide (NRP) or NRP-polyketide hybrid

Background: Plants produce antimicrobial phytochemicals that can reduce growth and infectivity of parasites in animals. Pollinator parasites are transmitted between hosts that forage on shared flowers. Floral transmission directly exposes parasites to phytochemicals on floral surfaces and in nectar, both at flowers and, post-ingestion, in the crop. This exposure could directly affect parasite transmission to new hosts. Approach: We combined nectar chemical analyses with field and cell culture experiments to test effects of the floral phytochemical thymol on transmission potential of the trypanosomatid gut parasite Crithidia in Bombus impatiens. First, we measured thymol concentrations in Thymus vulgaris nectar. Second, we tested how addition of thymol to floral nectaries affected parasite transmission to foraging bees. Third, we used cell cultures to determine direct, dose-dependent effects of shortterm thymol exposure on subsequent in vitro parasite growth. Results: We found 26.1 ppm thymol in Thymus vulgaris nectar, 5-fold higher than previously documented in this species. However, addition of thymol to flowers of parasite-inoculated inflorescences of four plant species did not affect acquisition of Crithidia infection during a foraging bout. Cell culture experiments showed that thymol concentrations needed to reduce subsequent Crithidia growth by 50% (120 ppm) were 4.6-fold higher than the highest detected nectar concentration. Conclusions: Although thymol exposure can influence Crithidia viability, Crithidia are robust to the duration and magnitude of exposure encountered during floral foraging under natural conditions. Our experiments suggest that any effects of thymol alone on Crithidia-host infection dynamics probably reflect indirect, possibly host-mediated, effects of chronic thymol ingestion.