Cellulamides: A New Family of Marine-Sourced Linear Peptides from the Underexplored Cellulosimicrobium Genus.

Bioprospecting the secondary metabolism of underexplored Actinomycetota taxa is a prolific route to uncover novel chemistry. In this work, we report the isolation, structure elucidation, and bioactivity screening of cellulamides A and B (1 and 2), two novel linear peptides obtained from the culture of the macroalga-associated Cellulosimicrobium funkei CT-R177. The host of this microorganism, the Chlorophyta Codium tomentosum, was collected in the northern Portuguese coast and, in the scope of a bioprospecting study focused on its associated actinobacterial community, strain CT-R177 was isolated, taxonomically identified, and screened for the production of antimicrobial and anticancer compounds. Dereplication of a crude extract of this strain using LC-HRMS(/MS) analysis unveiled a putative novel natural product, cellulamide A (1), that was isolated following mass spectrometry-guided fractionation. An additional analog, cellulamide B (2) was obtained during the chromatographic process and chemically characterized. The chemical structures of the novel linear peptides, including their absolute configurations, were elucidated using a combination of HRMS, 1D/2D NMR spectroscopy, and Marfey's analysis. Cellulamide A (1) was subjected to a set of bioactivity screenings, but no significant biological activity was observed. The cellulamides represent the first family of natural products reported from the Actinomycetota genus Cellulosimicrobium, showcasing not only the potential of less-explored taxa but also of host-associated marine strains for novel chemistry discovery.

Onychocolone A produced by the fungus Onychocola sp. targets cancer stem cells and stops pancreatic cancer progression by inhibiting MEK2-dependent cell signaling.

Pancreatic cancer (PC) shows a high fatality rate that can only be faced with a combination of surgery and chemotherapy or palliative treatment in the case of advanced patients. Besides, PC tumors are enriched with subpopulations of cancer stem cells (CSCs) that are resistant to the existing chemotherapeutic agents, which raises an important need for the identification of new drugs. To fill this gap, we have tested the anti-tumoral activity of microbial extracts, which chemical diversity offers a broad spectrum of potential new bioactive compounds. Extracts derived from the fungus Onychocola sp. CF-107644 were assayed via high throughput screening followed by bioassay-guided fractionation and resulted in the identification and isolation of six benzophenone derivatives with antitumoral activity: onychocolones A-F (#1-6). The structures of the compounds were established by spectroscopic methods, including ESI-TOF MS, 1D and 2D NMR analyses and X-ray diffraction. Compounds #1-4 significantly inhibited the growth of the pancreas tumoral cell lines, with low-micromolar Median Effective Doses (ED50s). Compound #1 (onychocolone A) was prioritized for further profiling due to its pro-apoptotic effect, which was further validated on 3D spheroids and pancreatic CSCs. Protein expression assays showed that the effect was mechanistically linked to the inhibition of MEK onco-signaling pathway. The efficacy of onychocolone A was also demonstrated in vivo by the reduction of tumor growth in a pancreatic xenograft mouse model generated by CSCs. Altogether, the data support that onychocolone A is a promising new small molecule for hit-to-lead development of a new treatment for PC.

Unearthing a Cryptic Biosynthetic Gene Cluster for the Piperazic Acid-Bearing Depsipeptide Diperamycin in the Ant-Dweller Streptomyces sp. CS113.

Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house "Carlos Sialer (CS)" strain collection consists of seventy-one Streptomyces strains isolated from the cuticle of leaf-cutting ants of the tribe Attini. Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC dpn in Streptomyces sp. CS113, which encodes the biosynthesis of the hybrid polyketide-depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the dpn BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene dpnS2 and the piperazate synthase gene dpnZ. The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the dpn BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis.

Delftia tsuruhatensis TC1 symbiont suppresses malaria transmission by anopheline mosquitoes.

Malaria control demands the development of a wide range of complementary strategies. We describe the properties of a naturally occurring, non-genetically modified symbiotic bacterium, Delftia tsuruhatensis TC1, which was isolated from mosquitoes incapable of sustaining the development of Plasmodium falciparum parasites. D. tsuruhatensis TC1 inhibits early stages of Plasmodium development and subsequent transmission by the Anopheles mosquito through secretion of a small-molecule inhibitor. We have identified this inhibitor to be the hydrophobic molecule harmane. We also found that, on mosquito contact, harmane penetrates the cuticle, inhibiting Plasmodium development. D. tsuruhatensis TC1 stably populates the mosquito gut, does not impose a fitness cost on the mosquito, and inhibits Plasmodium development for the mosquito's life. Contained field studies in Burkina Faso and modeling showed that D. tsuruhatensis TC1 has the potential to complement mosquito-targeted malaria transmission control.

Uncovering the Cryptic Gene Cluster ahb for 3-amino-4-hydroxybenzoate Derived Ahbamycins, by Searching SARP Regulator Encoding Genes in the Streptomyces argillaceus Genome.

Genome mining using standard bioinformatics tools has allowed for the uncovering of hidden biosynthesis gene clusters for specialized metabolites in Streptomyces genomes. In this work, we have used an alternative approach consisting in seeking "Streptomyces Antibiotic Regulatory Proteins" (SARP) encoding genes and analyzing their surrounding DNA region to unearth cryptic gene clusters that cannot be identified using standard bioinformatics tools. This strategy has allowed the unveiling of the new ahb cluster in Streptomyces argillaceus, which had not been retrieved before using antiSMASH. The ahb cluster is highly preserved in other Streptomyces strains, which suggests a role for their encoding compounds in specific environmental conditions. By combining overexpression of three regulatory genes and generation of different mutants, we were able to activate the ahb cluster, and to identify and chemically characterize the encoded compounds that we have named ahbamycins (AHBs). These constitute a new family of metabolites derived from 3-amino-4-hydroxybenzoate (3,4-AHBA) known for having antibiotic and antitumor activity. Additionally, by overexpressing three genes of the cluster (ahbH, ahbI, and ahbL2) for the synthesis and activation of 3,4-AHBA, a new hybrid compound, AHB18, was identified which had been produced from a metabolic crosstalk between the AHB and the argimycin P pathways. The identification of this new BGC opens the possibility to generate new compounds by combinatorial biosynthesis.

Combinatorial biosynthesis yields novel hybrid argimycin P alkaloids with diverse scaffolds in Streptomyces argillaceus.

Coelimycin P1 and argimycins P are two types of polyketide alkaloids produced by Streptomyces coelicolor and Streptomyces argillaceus, respectively. Their biosynthesis pathways share some early steps that render very similar aminated polyketide chains, diverging the pathways afterwards. By expressing the putative isomerase cpkE and/or the putative epoxidase/dehydrogenase cpkD from the coelimycin P1 gene cluster into S. argillaceus wild type and in argimycin mutant strains, five novel hybrid argimycins were generated. Chemical characterization of those compounds revealed that four of them show unprecedented scaffolds (quinolizidine and pyranopyridine) never found before in the argimycin family of compounds. One of these compounds (argimycin DM104) shows improved antibiotic activity. Noticeable, biosynthesis of these quinolizidine argimycins results from a hybrid pathway created by combining enzymes from two different pathways, which utilizes an aminated polyketide chain as precursor instead of lysine as it occurs for other quinolizidines.

Expanding the utility of Marfey's analysis by using HPLC-SPE-NMR to determine the Cß configuration of threonine and isoleucine residues in natural peptides.

The determination of amino acid chirality in natural peptides is typically addressed by Marfey's analysis. This approach relies on the complete hydrolysis of the peptide followed by the reaction of the resulting amino acid pool with Marfey's reagent, a chiral derivatizing agent which turns amino acid enantiomers into diastereomeric pairs which can be resolved by conventional reversed-phase HPLC. However, for certain amino acids possessing a second chiral centre at Cß, the discrimination between the two possible epimers may still be challenging due to the lack of chromatographic resolution. Such is the case of isoleucine and threonine which can also be found in natural nonribosomal peptides as their allo-diastereomers. We describe a new approach based on the extension of Marfey's analysis using HPLC-SPE-NMR to sort out this challenge. Marfey's derivatives of these epimeric amino acids at Cß can be differentiated by their distinct NMR spectra. Thus, simple comparison of the NMR spectra of trapped HPLC peaks with the corresponding spectra of standards enables the unambiguous assignment of the absolute configuration at the second chiral centre in such cases. The general applicability of this approach is showcased for two model cyclic peptides bearing L-Ile and L-Thr.

Biosynthesis of Largimycins in Streptomyces argillaceus Involves Transient ß-Alkylation and Cryptic Halogenation Steps Unprecedented in the Leinamycin Family.

Largimycins A1 and A2 are key members of a recently identified family of hybrid nonribosomal peptide polyketides belonging to the scarcely represented group of antitumor leinamycins. They are encoded by the gene cluster lrg of Streptomyces argillaceus. This cluster contains a halogenase gene and two sets of genes for the biosynthesis and incorporation of ß branches at C3 and C9. Noticeably, largimycins A1 and A2 are nonhalogenated compounds and only contain a ß branch at C3. By generating mutants in those genes and characterizing chemically their accumulated compounds, we could confirm the existence of a chlorination step at C19, the introduction of an acetyl-derived olefinic exomethylene group at C9, and a propionyl-derived ß branch at C3 in the biosynthesis pathway. Since the olefinic exomethylene group and the chlorine atom are absent in the final products, those biosynthetic steps can be considered cryptic in the overall pathway but essential to generating keto and epoxide functionalities at C9 and C18/C19, respectively. We propose that chlorination at C19 is utilized as an activation strategy that creates the precursor halohydrin to finally yield the epoxy functionality at C18/C19. This represents a novel strategy to create such functionalities and extends the small number of natural product biosynthetic pathways that include a cryptic chlorination step.

Curvicollide D Isolated from the Fungus Amesia sp. Kills African Trypanosomes by Inhibiting Transcription.

Sleeping sickness or African trypanosomiasis is a serious health concern with an added socio-economic impact in sub-Saharan Africa due to direct infection in both humans and their domestic livestock. There is no vaccine available against African trypanosomes and its treatment relies only on chemotherapy. Although the current drugs are effective, most of them are far from the modern concept of a drug in terms of toxicity, specificity and therapeutic regime. In a search for new molecules with trypanocidal activity, a high throughput screening of 2000 microbial extracts was performed. Fractionation of one of these extracts, belonging to a culture of the fungus Amesia sp., yielded a new member of the curvicollide family that has been designated as curvicollide D. The new compound showed an inhibitory concentration 50 (IC50) 16-fold lower in Trypanosoma brucei than in human cells. Moreover, it induced cell cycle arrest and disruption of the nucleolar structure. Finally, we showed that curvicollide D binds to DNA and inhibits transcription in African trypanosomes, resulting in cell death. These results constitute the first report on the activity and mode of action of a member of the curvicollide family in T. brucei.

Grapevine Xylem Sap Is a Potent Elicitor of Antibiotic Production in Streptomyces spp.

Streptomyces bacteria produce a wide number of antibiotics and antitumor compounds that have attracted the attention of pharmaceutical and biotech companies. In this study, we provide evidence showing that the xylem sap from grapevines has a positive effect on the production of different antibiotics by several Streptomyces species, including S. ambofaciens ATCC 23877 and S. argillaceus ATCC 12596 among others. The production of several already known compounds was induced: actinomycin D, chromomycin A3, fungichromin B, mithramycin A, etc., and four compounds with molecular formulas not included in the Dictionary of Natural Products (DNP v28.2) were also produced. The molecules present in the xylem sap that acts as elicitors were smaller than 3 kDa and soluble in water and insoluble in ether, ethyl acetate, or methanol. A combination of potassium citrate and di-D-fructose dianhydrides (related to levanbiose or inulobiose) seemed to be the main effectors identified from the active fraction. However, the level of induction obtained in the presence of these compounds mix was weaker and delayed with respect to the one got when using the whole xylem sap or the 3 kDa sap fraction, suggesting that another, not identified, elicitor must be also implied in this induction.

Euglenatides, Potent Antiproliferative Cyclic Peptides Isolated from the Freshwater Photosynthetic Microalga Euglena gracilis.

By limiting the nitrogen source to glutamic acid, we isolated cyclic peptides from Euglena gracilis containing asparagine and non-proteinogenic amino acids. Structure elucidation was accomplished through spectroscopic methods, mass spectrometry and chemical degradation. The euglenatides potently inhibit pathogenic fungi and cancer cell lines e.g., euglenatide B exhibiting IC50 values of 4.3 µM in Aspergillus fumigatus and 0.29 µM in MCF-7 breast cancer cells. In an unprecedented convergence of non-ribosomal peptide synthetase and polyketide synthase assembly-line biosynthesis between unicellular species and the metazoan kingdom, euglenatides bear resemblance to nemamides from Caenorhabditis elegans and inhibited both producing organisms E. gracilis and C. elegans. By molecular network analysis, we detected over forty euglenatide-like metabolites in E. gracilis, E. sanguinea and E. mutabilis, suggesting an important biological role for these natural products.

Colibrimycins, Novel Halogenated Hybrid Polyketide Synthase-Nonribosomal Peptide Synthetase (PKS-NRPS) Compounds Produced by Streptomyces sp. Strain CS147.

The improvement of genome sequencing techniques has brought to light the biosynthetic potential of actinomycetes due to the large number of gene clusters they present compared to the number of known compounds. Genome mining is a recent strategy in the search for novel bioactive compounds, which involves the analysis of sequenced genomes to identify uncharacterized natural product biosynthetic gene clusters, many of which are cryptic or silent under laboratory conditions, and to develop experimental approaches to identify their products. Owing to the importance of halogenation in terms of structural diversity, bioavailability, and bioactivity, searching for new halogenated bioactive compounds has become an interesting issue in the field of natural product discovery. Following this purpose, a screening for halogenase coding genes was performed on 12 Streptomyces strains isolated from fungus-growing ants of the Attini tribe. Using the bioinformatics tools antiSMASH and BLAST, six halogenase coding genes were identified. Some of these genes were located within biosynthetic gene clusters (BGCs), which were studied by construction of several mutants for the identification of the putative halogenated compounds produced. The comparison of the metabolite production profile of wild-type strains and their corresponding mutants by ultrahigh-performance liquid chromatography-UV and high-performance liquid chromatography-mass spectrometry allowed us the identification of a novel family of halogenated compounds in Streptomyces sp. strain CS147, designated colibrimycins. IMPORTANCE Genome mining has proven its usefulness in the search for novel bioactive compounds produced by microorganisms, and halogenases comprise an interesting starting point. In this work, we have identified a new halogenase coding gene that led to the discovery of novel lipopetide nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS)-derived natural products, the colibrimycins, produced by Streptomyces sp. strain CS147, isolated from the Attini ant niche. Some colibrimycins display an unusual α-ketoamide moiety in the peptide structure. Although its biosynthetic origin remains unknown, its presence might be related to a hypothetical inhibition of virus proteases, and, together with the presence of the halogenase, it represents a feature to be incorporated in the arsenal of structural modifications available for combinatorial biosynthesis.

Caerulines A and B, Flavonol Diacylglycosides from Persea caerulea.

Two undescribed 4'-O-methylkaempferol-[3″,4″-di-p-coumaroyl]-α-l-rhamnopyranosides, caerulines A and B (1-2), along with three known 4'-O-methylkaempferol diacylrhamnosides isomers (3-5) were isolated from an ethanol extract of the leaves of Persea caerulea, a native plant growing on the Colombian Caribbean coast. The chemical structures of 1 and 2 were elucidated by spectroscopic methods. The effect of compounds 1-5 against four pathogenic microorganisms [i.e., methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Candida albicans, and Aspergillus fumigatus] was tested in vitro. The compounds exhibited no activity against these pathogens except MRSA (MIC 12-48 µg/mL). Caeruline B (2) was found to be the most active compound with a modest anti-MRSA activity (MIC = 12 µg/mL).

Preclinical evaluation of strasseriolides A-D, potent antiplasmodial macrolides isolated from Strasseria geniculata CF-247,251.

BACKGROUND: Malaria is a global health problem for which novel therapeutic compounds are needed. To this end, a recently published novel family of antiplasmodial macrolides, strasseriolides A-D, was herein subjected to in vivo efficacy studies and preclinical evaluation in order to identify the most promising candidate(s) for further development. METHODS: Preclinical evaluation of strasseriolides A-D was performed by MTT-based cytotoxicity assay in THLE-2 (CRL-2706) liver cells, cardiotoxicity screening using the FluxOR™ potassium assay in hERG expressed HEK cells, LC-MS-based analysis of drug-drug interaction involving CYP3A4, CYP2D6 and CYP2C9 isoforms inhibition and metabolic stability assays in human liver microsomes. Mice in vivo toxicity studies were also accomplished by i.v. administration of the compounds (vehicle: 0.5% HPMC, 0.5% Tween 80, 0.5% Benzyl alcohol) in mice at 25 mg/kg dosage. Plasma were prepared from mice blood samples obtained at different time points (over a 24-h period), and analysed by LC-MS to quantify compounds. The most promising compounds, strasseriolides C and D, were subjected to a preliminary in vivo efficacy study in which transgenic GFP-luciferase expressing Plasmodium berghei strain ANKA-infected Swiss Webster female mice (n = 4-5) were treated 48 h post-infection with an i.p. dosage of strasseriolide C at 50 mg/kg and strasseriolide D at 22 mg/kg for four days after which luciferase activity was quantified on day 5 in an IVIS® Lumina II imager. RESULTS: Strasseriolides A-D showed no cytotoxicity, no carditoxicity and no drug-drug interaction problems in vitro with varying intrinsic clearance (CLint). Only strasseriolide B was highly toxic to mice in vivo (even at 1 mg/kg i.v. dosage) and, therefore, discontinued in further in vivo studies. Strasseriolide D showed statistically significant activity in vivo giving rise to lower parasitaemia levels (70% lower) compared to the controls treated with vehicle. CONCLUSIONS: Animal efficacy and preclinical evaluation of the recently discovered potent antiplasmodial macrolides, strasseriolides A-D, led to the identification of strasseriolide D as the most promising compound for further development. Future studies dealing on structure optimization, formulation and establishment of optimal in vivo dosage explorations of this novel compound class could enhance their clinical potency and allow for progress to later stages of the developmental pipeline.

Anti-CD44-Conjugated Olive Oil Liquid Nanocapsules for Targeting Pancreatic Cancer Stem Cells.

The latest trends in cancer research and nanomedicine focus on using nanocarriers to target cancer stem cells (CSCs). Specifically, lipid liquid nanocapsules are usually developed as nanocarriers for lipophilic drug delivery. Here, we developed olive oil liquid NCs (O2LNCs) functionalized by covalent coupling of an anti-CD44-fluorescein isothiocyanate antibody (αCD44). First, O2LNCs are formed by a core of olive oil surrounded by a shell containing phospholipids, a nonionic surfactant, and deoxycholic acid molecules. Then, O2LNCs were coated with an αCD44 antibody (αCD44-O2LNC). The optimization of an αCD44 coating procedure, a complete physicochemical characterization, as well as clear evidence of their efficacy in vitro and in vivo were demonstrated. Our results indicate the high targeted uptake of these αCD44-O2LNCs, and the increased antitumor efficacy (up to four times) of paclitaxel-loaded-αCD44-O2LNC compared to free paclitaxel in pancreatic CSCs (PCSCs). Also, αCD44-O2LNCs were able to selectively target PCSCs in an orthotopic xenotransplant in vivo model.

Co-occurring Congeners Reveal the Position of Enantiomeric Amino Acids in Nonribosomal Peptides.

The absolute configuration of the constituent amino acids in microbial nonribosomal peptides is typically determined by Marfey's method after total hydrolysis of the peptide. A challenge to structure elucidation arises when both d and l enantiomeric configurations of an amino acid are present. Determining the actual position of each amino acid enantiomer within the peptide sequence typically requires laborious approaches based on peptide partial hydrolysis or even total synthesis of the possible diastereomers. Herein, an alternative solution is discussed based on the homogeneous backbone chirality that governs all peptides biosynthesized by a common nonribosomal peptide synthetase. The information on configuration provided by Marfey's analysis of co-occurring minor congeners can reveal unequivocally the stereochemical sequence of the whole peptide family.

Strasseriolides A-D, A Family of Antiplasmodial Macrolides Isolated from the Fungus Strasseria geniculata CF-247251.

A novel family of four potent antimalarial macrolides, strasseriolides A-D (1-4), has been isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. The structures of these compounds, including their absolute configurations, were elucidated by HRMS, NMR spectroscopy, and X-ray single-crystal diffraction. The four compounds gave respective IC50 values of 9.810, 0.013, 0.123, and 0.128 µM against Plasmodium falciparum 3D7 parasites with no significant cytotoxicity against the HepG2 cell line.

Viennamycins: Lipopeptides Produced by a Streptomyces sp.

Extracts from Streptomyces sp. S4.7 isolated from the rhizosphere of edelweiss, an alpine medicinal plant, exhibited activity against Gram-positive bacteria. LC-HRMS analyses of the extracts resulted in the detection of two unknown, structurally related lipopeptides that were assumed to be responsible for the antibiotic activity. LC-MS guided isolation and structure elucidation of viennamycins A and B (1 and 2) by HR-MS/MS, 1D and 2D NMR, and Marfey's analyses revealed them to be novel compounds, with viennamycin A containing cysteic acid, a unique feature for lipopeptides. Tests for antibacterial, antifungal, and cytotoxic activities of purified viennamycins, both with and without divalent cations, did not reveal any bioactivity, suggesting that their biological function, which could not be determined in the tests used, is atypical for lipopeptides. The genome of Streptomyces sp. S4.7 was sequenced and analyzed, revealing the viennamycin biosynthetic gene cluster. Detailed bioinformatics-based analysis of the viennamycin gene cluster allowed elucidation of the biosynthetic pathway for these lipopeptides.

Structural Elucidation of Antibiotic TKR2999, an Antifungal Lipodepsipeptide Isolated from the Fungus Foliophoma fallens.

An antifungal lipodepsipeptide was obtained from cultures of the fungus Foliophoma fallens CF-236885. Its structure, elucidated by HRMS and NMR spectroscopy, contained Gly, Thr, Asn, ß-Ala, Orn, Ala, two Ser residues, and 3-hydroxy-4-methylhexadecanoic acid. The absolute configuration of its amino acid residues was determined using Marfey's analysis and J-based configuration analysis helped to establish the relative configuration of the 3-hydroxy-4-methylhexadecanoic acid moiety. A literature search retrieved a patent describing antibiotic TKR2999 (1), whose non-disclosed structure was confirmed to be identical to that found for our compound, according to its physicochemical properties and NMR spectra. Compound 1 displayed potent antifungal activity against Aspergillus fumigatus and a panel of Candida strains.

Discovery of Cryptic Largimycins in Streptomyces Reveals Novel Biosynthetic Avenues Enriching the Structural Diversity of the Leinamycin Family.

Largimycins are hybrid nonribosomal peptide-polyketides that constitute a new group of metabolites in the leinamycin family of natural products displaying unique structural features such as containing an oxazole instead of a thiazole ring or being oxime ester macrocycles, unprecedented in nature, rather than macrolactams. Their discovery in Streptomyces argillaceus and Streptomyces canus has relied on the activation of two homologous silent gene clusters by overexpressing a transcriptional activator and cultivating in specific media. The proposed biosynthesis of largimycins includes the key action of the oxidoreductase LrgO, responsible for the formation of the oxime group involved in macrocyclization, and two putative cryptic biosynthetic steps consisting of chlorination of l-Thr by the NRPS loading module and incorporation of an olefinic exomethylene group by LrgJ PKS. The discovery of largimycins uncovers novel biosynthetic avenues employed in nature to enrich the structural diversity of leinamycins and provides tools for combinatorial biosynthesis.