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We describe activity-based protein profiling for analyzing the adenylation domains of non-ribosomal peptide synthetases (ABPP-NRPS) in bacterial proteomes. Using a range of non-proteoinogenic amino acid sulfamoyladenosines, the competitive format of ABPP-NRPS provided substrate tolerance toward non-proteinogenic amino acids. When coupled with precursor-directed biosynthesis, a non-proteinogenic amino acid ( O -allyl-L-serine) was successfully incorporated into gramicidin S.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) with polar-functionalized fatty acyl groups are newly found lipopeptide-class natural products. We recently employed a combined approach of genome mining and stable isotope labeling and discovered solabiomycins as one of the polar-functionalized fatty-acylated RiPPs (PFARs) from Streptomyces lydicus NBRC13058. The solabiomycins contained a characteristic sulfoxide group in the labionin moiety referred to as the 'solabionin' structure for the RiPP moiety. A previous gene knockout experiment indicated that solS, which encodes a putative flavin adenine dinucleotide (FAD)-nicotinamide adenine dinucleotide (phosphate) (NAD(P))-binding protein, is involved in the sulfoxidation of an alkyl sulfide in the solabionin. In this study, we isolated deoxysolabiomycins A and B from ΔsolS mutant and fully determined the chemical structures using a series of NMR experiments. We also tested the bioactivity of deoxysolabiomycins against Gram-positive bacteria, including Mycolicibacterium smegmatis, and notably found that the sulfoxide is critical for the antibacterial activity. To characterize the catalytic activity of SolS, the recombinant protein was incubated with a putative substrate, deoxysolabiomycins, and the cofactors FAD and NADPH. In vitro reactions demonstrated that SolS catalyzes the sulfoxidation, converting deoxysolabiomycins to solabiomycins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Longicatenamides A-D, two diastereomeric pairs of new cyclic hexapeptides, were isolated from the combined-culture of Streptomyces sp. KUSC_F05 and Tsukamurella pulmonis TP-B0596. Their planar structures were determined by spectroscopic analysis including extensive 2D NMR and MS analysis. The absolute configurations of their component amino acids were determined by the use of highly sensitive reagents we recently developed; the highly sensitive-advanced Marfey's method (HS-advanced Marfey's method), which led us to reduce the sample loss and prevent incorrect structural determination. Particularly, the C β -stereochemistry of hyGlu in longicatenamides A and C was assigned without any use of C β -Marfey's methods. Longicatenamide A exhibited weak but preferential antimicrobial activity against Bacillus subtilis.

Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, we establish a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis reveals remarkable substrate tolerance of the biosynthetic enzymes and leads to the development of the minimal lactazole scaffold, a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz opens access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and 18 amino acid-long tail regions, as well as to hybrid thiopeptides containing non-proteinogenic amino acids. This work suggests that the minimal lactazole scaffold is amenable to extensive bioengineering and opens possibilities to explore untapped chemical space of thiopeptides.

The production of two new heterocyclic peptide isomers, catenulobactins A (1) and B (2), in cultures of Catenuloplanes sp. RD067331 was significantly increased when it was cocultured with a mycolic acid-containing bacterium. The planar structures and absolute configurations of the catenulobactins were determined based on NMR/MS and chiral-phase GC-MS analyses. Catenulobactin B (2) displayed Fe(III)-chelating activity and moderate cytotoxicity against P388 murine leukemia cells.

Goadsporin (GS) is a member of ribosomally synthesized and post-translationally modified peptides (RiPPs), containing an N-terminal acetyl moiety, six azoles and two dehydroalanines in the peptidic main chain. Although the enzymes involved in GS biosynthesis have been defined, the principle of how the respective enzymes control the specific modifications remains elusive. Here we report a one-pot synthesis of GS using the enzymes reconstituted in the 'flexible' in vitro translation system, referred to as the FIT-GS system. This system allows us to readily prepare not only the precursor peptide from its synthetic DNA template but also 52 mutants, enabling us to dissect the modification determinants of GodA for each enzyme. The in vitro knowledge has also led us to successfully produce designer GS analogues in vivo. The methodology demonstrated in this work is also applicable to other RiPP biosynthesis, allowing us to rapidly investigate the principle of modification events with great ease., Competing Interests: The authors declare no competing financial interests.

Dehydroalanines in goadsporin are proposed to be formed by GodF and GodG, which show slight homology to the N-terminal glutamylation and C-terminal elimination domains, respectively, of LanB, a class I lanthipeptide dehydratase. Although similar, separated-type LanBs are conserved among thiopeptides and indispensable for their biosynthesis and biological activities, these enzymes had not yet been characterized. Here, we identified goadsporin B, which has unmodified Ser4 and Ser14, from both godF and godG disruptants. The godG disruptant also produced goadsporin C, a glutamylated-Ser4 variant of goadsporin B. These results suggested that dehydroalanines are formed by glutamylation and glutamate elimination. NMR analysis revealed for the first time that the glutamyl group was attached to a serine via an ester bond, by the catalysis of LanB-type enzymes. Our findings provide insights into the function of separated-type LanBs involved in the biosynthesis of goadsporin and thiopeptides., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although >100 thiopeptides have been discovered, the number of validated gene clusters involved in their biosynthesis is lagging. We use genome mining to identify a silent thiopeptide biosynthetic gene cluster responsible for biosynthesis of lactazoles. Lactazoles are structurally unique thiopeptides with a 32-membered macrocycle and a 2-oxazolyl-6-thiazolyl pyridine core. We demonstrate that lactazoles originate from the simplest cluster, containing only six unidirectional genes (lazA to lazF). We show that lazC is involved in the macrocyclization process, leading to central pyridine moiety formation. Substitution of the endogenous promoter with a strong promoter results in an approximately 30-fold increase in lactazole A production and mutagenesis of lazC precursor gene in production of two analogs. Lactazoles do not exhibit antimicrobial activity but may modulate signaling cascades triggered by bone morphogenetic protein. Our approach facilitates the production of a more diverse set of thiopeptide structures, increasing the semisynthetic repertoire for use in drug development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Improving the productivity of secondary metabolites is highly beneficial for the utilization of natural products. Here, we found that gene duplication of the goadsporin biosynthetic gene locus resulted in hyper-production. Goadsporin is a linear azole containing peptide that is biosynthesized via a ribosome-mediated pathway in Streptomyces sp. TP-A0584. Recombinant strains containing duplicated or triplicated goadsporin biosynthetic gene clusters produced 1.46- and 2.25-fold more goadsporin than the wild-type strain. In a surrogate host, Streptomyces lividans, chromosomal integration of one or two copies of the gene cluster led to 342.7 and 593.5 mg/L of goadsporin production. Expression of godI, a self-resistance gene, and of godR, a pathway-specific transcriptional regulator, under a constitutive promoter gave 0.79- and 2.12-fold higher goadsporin production than the wild-type strain. Our experiments indicated that a proportional relationship exists between goadsporin production per culture volume and the copy number of the biosynthetic gene cluster.

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

The biosynthesis of staurosporine, rebeccamycin, and goadsporin, which are produced by actinomycetes and contain characteristic heterocyclic rings, was characterized by genetic methods. Staurosporine and rebeccamycin contain an indolocarbazole ring synthesized from two molecules of tryptophan, with indolepyruvic acid imine and chromopyrrolic acid as biosynthetic intermediates. A tetrameric hemoprotein synthesizes chromopyrrolic acid, and cytochrome P450 peroxidase catalyzes the intramolecular C-C coupling and decarboxylation of chromopyrrolic acid to form the indolocarbazole core. Goadsporin is a thiopeptide containing thiazole and oxazole heterocyclic rings. The structural gene godA is ribosomally translated to a goadsporin precursor peptide, and oxazole, methyloxazole, and thiazole rings are derived from serine, threonine, and cystein through post-translational modifications. On the basis of these knowledges, a wide variety of indolocarbazole and goadsporin analogs through the rational gene recombination and disruption of these biosynthetic genes were successfully produced.

The biosynthetic gene cluster of goadsporin, a polypeptide antibiotic containing thiazole and oxazole rings, was cloned from Streptomyces sp. TP-A0584. The cluster contains a structural gene, godA, and nine god (goadsporin) genes involved in post-translational modification, immunity and transcriptional regulation. Although the gene organization is similar to typical bacteriocin biosynthetic gene clusters, each goadsporin biosynthetic gene shows low homology to these genes. Goadsporin biosynthesis is initiated by the translation of godA, and the subsequent cyclization, dehydration and acetylation are probably catalysed by godD, godE, godF, godG and godH gene products. godI shows high similarity to the 54 kDa subunit of the signal recognition particle and plays an important role in goadsporin immunity. Furthermore, four goadsporin analogues were produced by site-directed mutagenesis of godA, suggesting that this biosynthesis machinery is used for the heterocyclization of peptides.