The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, which are proposed to be introduced by YcaO-TfuA proteins. Using RiPPER, we show that previously undescribed RiPP gene clusters encoding YcaO and TfuA proteins are widespread in Actinobacteria and encode a highly diverse landscape of precursor peptides that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new structural class of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis .

Bottromycin A2 is a structurally unique ribosomally synthesized and post‐translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrug‐resistant bacteria. The structural novelty of bottromycin stems from its unprecedented macrocyclic amidine and rare β‐methylated amino acid residues. The N‐terminus of a precursor peptide (BtmD) is converted into bottromycin A2 by tailoring enzymes encoded in the btm gene cluster. However, little was known about key transformations in this pathway, including the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approach that harnesses mass spectral networking was used to assess the metabolomes of a series of pathway mutants. This analysis has yielded key information on the function of a variety of previously uncharacterized biosynthetic enzymes, including a YcaO domain protein and a partner protein that together catalyze the macrocyclization.

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Bottromycin A 2 is astructurally unique ribosomally synthesized and post-translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrugresistant bacteria. The structural noveltyofbottromycin stems from its unprecedented macrocyclic amidine and rare bmethylated amino acid residues.The N-terminus of aprecursor peptide (BtmD) is converted into bottromycin A 2 by tailoring enzymes encoded in the btm gene cluster.H owever,l ittle was knowna bout key transformations in this pathway,i ncluding the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approacht hat harnesses mass spectral networking was used to assess the metabolomes of aseries of pathway mutants.This analysis has yielded key information on the function of av ariety of previously uncharacterized biosynthetic enzymes,i ncluding aY caO domain protein and ap artner protein that together catalyze the macrocyclization.Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products that are prevalent throughout nature, [1] and their biosynthetic pathways are capable of transforming simple proteinogenic amino acids into structurally complex compounds that have potent bioactivities. [2][3][4] However,e lucidating the biosynthesis of RiPPs can be hindered by the difficulty of isolating intermediates,asthe biosynthesis takes place on alarger precursor peptide,a nd intermediates may be rapidly proteolyzed. Therefore,i mproved methods for the identification of RiPP intermediates are desirable.Bottromycin A 2 (1,Scheme 1) [5][6][7][8] possesses potent antibacterial activity towards multidrugresistant bacteria, [9] and is structurally unique owing its unprecedented macrocyclic amidine,r are b-methylated amino acids residues,and aterminal thiazole.Nature employs av ariety of strategies for peptide macrocyclization, [10][11][12] but amidine formation has only been observed for bottromycin. Initial studies on bottromycin biosynthesis showed that its amino acids were b-methylated by radical SAM methyltransferases [5,7] (RSMTs), but the rest of the bottromycin pathway represented abiosynthetic black box, where little was known about key steps in the pathway,including the unprecedented macrocyclization. In this study,weemploy untargeted metabolomics and mass spectral networking to deduce the biosynthetic route to bottromycins in Streptomyces scabies.T his analysis identifies the enzymes responsible for macrocyclization, thiazole formation, and aspartate epimerization, thereby demonstrating the utility of an untargeted metabolomic approach for elucidating atargeted biosynthetic pathway.To assess the role of the putative tailoring genes in the bottromycin pathway (Supporting Information, Figure S1), we had previously generated S. scabies DbtmC, DbtmE, DbtmF, DbtmI,a nd DbtmJ,b ut were unable to identify bottromycin-like compounds in these mutants. [5] We therefore established that these deletions did not lead to deleterious polar effects on the pathway b...

Collismycin A is a member of the 2,2'-bipyridyl family of natural products that shows cytotoxic activity. Structurally, it belongs to the hybrid polyketides-nonribosomal peptides. After the isolation and characterization of the collismycin A gene cluster, we have used the combination of two different approaches (insertional inactivation and biocatalysis) to increase structural diversity in this natural product class. Twelve collismycin analogs were generated with modifications in the second pyridine ring of collismycin A, thus potentially maintaining biologic activity. None of these analogs showed better cytotoxic activity than the parental collismycin. However, some analogs showed neuroprotective activity and one of them (collismycin H) showed better values for neuroprotection against oxidative stress in a zebrafish model than those of collismycin A. Interestingly, this analog also showed very poor cytotoxic activity, a feature very desirable for a neuroprotectant compound.

Bicyclomycin (BCM) is a clinically promising antibiotic that is biosynthesized by Streptomyces cinnamoneus DSM 41675. BCM is structurally characterized by a core cyclo(l-Ile-l-Leu) 2,5-diketopiperazine (DKP) that is extensively oxidized. Here, we identify the BCM biosynthetic gene cluster, which shows that the core of BCM is biosynthesized by a cyclodipeptide synthase, and the oxidative modifications are introduced by five 2-oxoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase. The discovery of the gene cluster enabled the identification of BCM pathways encoded by the genomes of hundreds of Pseudomonas aeruginosa isolates distributed globally, and heterologous expression of the pathway from P. aeruginosa SCV20265 demonstrated that the product is chemically identical to BCM produced by S. cinnamoneus. Overall, putative BCM gene clusters have been found in at least seven genera spanning Actinobacteria and Proteobacteria (Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria). This represents a rare example of horizontal gene transfer of an intact biosynthetic gene cluster across such distantly related bacteria, and we show that these gene clusters are almost always associated with mobile genetic elements.IMPORTANCE Bicyclomycin is the only natural product antibiotic that selectively inhibits the transcription termination factor Rho. This mechanism of action, combined with its proven biological safety and its activity against clinically relevant Gram-negative bacterial pathogens, makes it a very promising antibiotic candidate. Here, we report the identification of the bicyclomycin biosynthetic gene cluster in the known bicyclomycin-producing organism Streptomyces cinnamoneus, which will enable the engineered production of new bicyclomycin derivatives. The identification of this gene cluster also led to the discovery of hundreds of bicyclomycin pathways encoded in highly diverse bacteria, including in the opportunistic pathogen Pseudomonas aeruginosa. This wide distribution of a complex biosynthetic pathway is very unusual and provides an insight into how a pathway for an antibiotic can be transferred between diverse bacteria.

Heterologous expression of biosynthetic gene clusters (BGCs) represents an attractive route to the production of new natural products, but is often hampered by poor yields. It is therefore important to develop tools that enable rapid refactoring, gene insertion/deletion, and targeted mutations in BGCs. Ideally, these tools should be highly efficient, affordable, accessible, marker free, and flexible for use with a wide range of BGCs. Here, we present a one-step yeast-based method that enables efficient, cheap, and flexible modifications to BGCs. Using the BGC for the antibiotic bottromycin, we showcase multiple modifications including refactoring, gene deletions and targeted mutations. This facilitated the construction of an inducible, riboswitch-controlled pathway that achieved a 120-fold increase in pathway productivity in a heterologous streptomycete host. Additionally, an unexpected biosynthetic bottleneck resulted in the production of a suite of new bottromycin-related metabolites.

Thiostreptamide S4 is a thioamitide, a family of promising antitumour ribosomally synthesised and post-translationally modified peptides (RiPPs). The thioamitides are one of the most structurally complex RiPP families, yet very...