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Microbial natural products form the basis of most of the antibiotics used in the clinic. The vast majority has not yet been discovered, among others because the hidden chemical space is obscured by previously identified (and typically abundant) antibiotics in culture extracts. Efficient dereplication is therefore key to the discovery of our future medicines. Here we present an analytical platform for the efficient identification and prioritization of low abundance bioactive compounds at nanoliter scale, called nanoRAPIDS. NanoRAPIDS encompasses analytical scale separation and nanofractionation of natural extracts, followed by the bioassay of interest, automated mass spectrometry identification, and Global Natural Products Social molecular networking (GNPS) for dereplication. As little as 10 μL crude extract is fractionated into 384 fractions. First, bioactive congeners of iturins and surfactins were identified in Bacillus, based on their bioactivity. Subsequently, bioactive molecules were identified in an extensive network of angucyclines elicited by catechol in cultures of Streptomyces sp. This allowed the discovery of a highly unusual N-acetylcysteine conjugate of saquayamycin, despite low production levels in an otherwise abundant molecular family. These data underline the utility and broad application of the technology for the prioritization of minor bioactive compounds in complex extracts. Microbial natural products are an important source for antibiotic discovery, however, their efficient dereplication remains challenging. Here, the authors develop an analytical pipeline, nanoRAPIDS, to prioritize low abundance bioactive compounds at nanoscale, by integrating the bioassay of interest, automated mass spectrometry identification and GNPS-based dereplication, resulting in the discovery of saquayamycin N from Streptomyces sp. MBT84. [ABSTRACT FROM AUTHOR]

Angucyclines are type II polyketide natural products, often characterized by unusual structural rearrangements through B- or C-ring cleavage of their tetracyclic backbone. While the enzymes involved in B-ring cleavage have been extensively studied, little is known of the enzymes leading to C-ring cleavage. Here, we unravel the function of the oxygenases involved in the biosynthesis of lugdunomycin, a highly rearranged C-ring cleaved angucycline derivative. Targeted deletion of the oxygenase genes, in combination with molecular networking and structural elucidation, showed that LugOI is essential for C12 oxidation and maintaining a keto group at C6 that is reduced by LugOII, resulting in a key intermediate towards C-ring cleavage. An epoxide group is then inserted by LugOIII, and stabilized by the novel enzyme LugOV for the subsequent cleavage. Thus, for the first time we describe the oxidative enzymatic steps that form the basis for a wide range of rearranged angucycline natural products. Angucyclines are a class of natural products that harbor unusual structural rearrangements through B- or C-ring cleavage of their tetracyclic backbone, however, the enzymes leading to C-ring cleavage remain poorly understood. Here, the authors use targeted gene deletion and complementation as well as metabolomics to study the function of putative oxygenases involved in lugdunomycin biosynthesis, and reveal their potential roles towards C-ring cleavage. [ABSTRACT FROM AUTHOR]

Streptomycetes are major producers of bioactive natural products, including the majority of the naturally produced antibiotics. While much of the low-hanging fruit has been discovered, it is predicted that less than 5% of the chemical space of natural products has been mined. Here, we describe the discovery of the novel actinomycins L1 and L2 produced by Streptomyces sp. MBT27, via application of metabolic analysis and molecular networking. Actinomycins L1 and L2 are diastereomers, and the structure of actinomycin L2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X2, prior to the condensation of the actinomycin halves. Such a structural feature has not previously been identified in naturally occurring actinomycins. Adding anthranilamide to cultures of the actinomycin X2 producer Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27, resulted in the production of actinomycin L. This supports a biosynthetic pathway whereby actinomycin L is produced from two distinct metabolic routes, namely those for actinomycin X2 and for anthranilamide. Actinomycins L1 and L2 showed significant antimicrobial activity against Gram-positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families. [ABSTRACT FROM AUTHOR]

Actinobacteria are a rich source of bioactive molecules, and genome sequencing has shown that the vast majority of their biosynthetic potential has yet to be explored. However, many of their biosynthetic gene clusters (BGCs) are poorly expressed in the laboratory, which prevents discovery of their cognate natural products. To exploit their full biosynthetic potential, better understanding of the signals that promote the expression of BGCs is needed. Here, we show that the human stress hormone epinephrine (adrenaline) elicits siderophore production by Actinobacteria. Catechol was established as the likely eliciting moiety, since similar responses were seen for catechol and for the catechol-containing molecules dopamine and catechin but not for related molecules. Exploration of the catechol-responsive strain Streptomyces sp. MBT84 using mass spectral networking revealed elicitation of a BGC that produces the angucycline glycosides aquayamycin, urdamycinone B and galtamycin C. Heterologous expression of the catechol-cleaving enzymes catechol 1,2-dioxygenase or catechol 2,3-dioxygenase counteracted the eliciting effect of catechol. Thus, our work identifies the ubiquitous catechol moiety as a novel elicitor of the expression of BGCs for specialized metabolites. The biosynthetic gene clusters of Actinobacteria are poorly expressed in the laboratory, necessitating a better understanding of the signals that promote their expression in order to exploit their full biosynthetic potential. Here, the authors show that the stress hormone epinephrine elicits the expression of biosynthetic gene clusters and influences the metabolism of Streptomyces, with the catechol moiety proving key to this response. [ABSTRACT FROM AUTHOR]