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.

Cytotoxic Activity of Saponins and Sapogenins Isolated from Chenopodium quinoa Willd. in Cancer Cell Lines.

The cytotoxic properties of two extracts from Chenopodium quinoa Willd. and three synthetic sapogenins were evaluated in different cancer cell lines (A549, SH-SY5Y, HepG2, and HeLa) to investigate their cytotoxic effects and determine if these cell lines activate the caspase pathway for apoptosis in response to saponin and sapogenin treatment. The saponin extracts were isolated from the agro-industrial waste of Chenopodium quinoa Willd., while the sapogenins were identified and quantitatively determined by High-Performance Liquid Chromatography (HPLC). Among these compounds, ursolic acid was the most active compound, with high IC50 values measured in all cell lines. In addition, hederagenin demonstrated higher caspase-3 activity than staurosporine in HeLa cells, suggesting an anti-cytotoxic activity via a caspase-dependent apoptosis pathway. HPLC analysis showed that the concentration of hederagenin was higher than that of oleanolic acid in ethanolic extracts of white and red quinoa. The ethanolic extracts of white and red quinoa did not show cytotoxic activity. On the other hand, the synthetic sapogenins such as ursolic acid, oleanolic acid, and hederagenin significantly decreased the viability of the four cell lines studied. Finally, by Caspase-3 assay, it was found that HeLa undergoes apoptosis during cell death because hederagenin produces a significant increase in PARP-1 hydrolysis in HeLa cells.

Identification of the Biosynthetic Gene Cluster of New Piperazic Acid-Containing Lipopeptides with Cytotoxic Activity in the Genome of Marine Streptomyces PHM034.

Three novel lipopeptides, PM130391 (1), PM130392 (2), and PM140293 (3) were obtained from cultures of Streptomyces tuirus PHM034 isolated from a marine sediment. Structural elucidation of the three compounds showed they belong to the nonribosomal peptides family, and they all contain an acylated alanine, three piperazic acids, a methylated glycine, and an N-hydroxylated alanine. The difference between the three compounds resides in the acyl chain bound to the alanine residue. All three compounds showed cytotoxic activity against human cancer cell lines. Genome sequence and bioinformatics analysis allowed the identification of the gene cluster responsible for the biosynthesis. Inactivation of a nonribosomal peptide synthase of this cluster abolished the biosynthesis of the three compounds, thus demonstrating the involvement of this cluster in the biosynthesis of these lipopeptides.

The Volatile Organic Compounds of Streptomyces spp.: An In-Depth Analysis of Their Antifungal Properties.

The study of volatile organic compounds (VOCs) has expanded because of the growing need to search for new bioactive compounds that could be used as therapeutic alternatives. These small molecules serve as signals to establish interactions with other nearby organisms in the environment. In this work, we evaluated the antifungal effect of VOCs produced by different Streptomyces spp. This study was performed using VOC chamber devices that allow for the free exchange of VOCs without physical contact between microorganisms or the diffusible compounds they produce. Antifungal activity was tested against Escovopsis weberi, a fungal pathogen that affects ant nest stability, and the results showed that Streptomyces spp. CS014, CS057, CS131, CS147, CS159, CS207, and CS227 inhibit or reduce the fungal growth with their emitted VOCs. A GS-MS analysis of volatiles produced and captured by activated charcoal suggested that these Streptomyces strains synthesize several antifungal VOCs, many of them produced because of the presence of E. weberi, with the accumulation of various VOCs determining the growth inhibition effect.

Co-Expression of Transcriptional Regulators and Housekeeping Genes in Streptomyces spp.: A Strategy to Optimize Metabolite Production.

The search for novel bioactive compounds to overcome resistance to current therapeutics has become of utmost importance. Streptomyces spp. are one of the main sources of bioactive compounds currently used in medicine. In this work, five different global transcriptional regulators and five housekeeping genes, known to induce the activation or overproduction of secondary metabolites in Streptomyces coelicolor, were cloned in two separated constructs and expressed in 12 different strains of Streptomyces spp. from the in-house CS collection. These recombinant plasmids were also inserted into streptomycin and rifampicin resistant Streptomyces strains (mutations known to enhance secondary metabolism in Streptomyces). Different media with diverse carbon and nitrogen sources were selected to assess the strains' metabolite production. Cultures were then extracted with different organic solvents and analysed to search for changes in their production profiles. An overproduction of metabolites already known to be produced by the biosynthesis wild-type strains was observed such as germicidin by CS113, collismycins by CS149 and CS014, or colibrimycins by CS147. Additionally, the activation of some compounds such as alteramides in CS090a pSETxkBMRRH and CS065a pSETxkDCABA or inhibition of the biosynthesis of chromomycins in CS065a in pSETxkDCABA when grown in SM10 was demonstrated. Therefore, these genetic constructs are a relatively simple tool to manipulate Streptomyces metabolism and explore their wide secondary metabolites production potential.

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.

In vitro release of aluminum from the geophagic clay "Chacco" in the Peruvian highlands: Chemical characterization and health risk assessment.

In the altiplano zone of Latin America, "Chacco" is one of the clays widely consumed as part of geophagy. The objectives of the study were to chemically characterize "Chacco", determine the zero charge point, evaluate the release of aluminum in vitro, perform the kinetic study and evaluate the health risk. The results by ICP-OES showed that the elements with the highest concentration were Al, Ba, Ca, Fe, K, Mg, Mn, Na, Si, Sr, Ti and Zn. ATR-FTIR analysis showed the presence of Si-O (693 and 990 cm-1), Al-O (790 cm-1), Al-Al-OH bending vibration (912 cm-1), Si-H bond stretching (2100 to 2500 cm-1) and free -OH groups (3629 cm-1). SEM-EDX results indicate that Al is one of the main constituents of "Chacco" (7.35 wt%). The pHzpc of "Chacco" was 6.83. In the dissolution profiles, the highest Al release occurred at pH 6.8 and in intestinal juice simulated with pseudo-second order dissolution kinetics. The EDIAl and EWIAl were 20.24 and 142.66 respectively, comparing EWIAl with the PTWI established by JECFA (2 mg/kg bw), it is concluded that the weekly intake of "Chacco" represents an appreciable health risk. There are no reports of the carcinogenic factor of Al, so TRAl was not calculated.

Volatile Compounds in Actinomycete Communities: A New Tool for Biosynthetic Gene Cluster Activation, Cooperative Growth Promotion, and Drug Discovery.

The increasing appearance of multiresistant pathogens, as well as emerging diseases, has highlighted the need for new strategies to discover natural compounds that can be used as therapeutic alternatives, especially in the genus Streptomyces, which is one of the largest producers of bioactive metabolites. In recent years, the study of volatile compounds (VOCs) has raised interest because of the variety of their biological properties in addition to their involvement in cell communication. In this work, we analyze the implications of VOCs as mediating molecules capable of inducing the activation of biosynthetic pathways of bioactive compounds in surrounding Actinomycetes. For this purpose, several strains of Streptomyces were co-cultured in chamber devices that allowed VOC exchange while avoiding physical contact. In several of those strains, secondary metabolism was activated by VOCs emitted by companion strains, resulting in increased antibiotic production and synthesis of new VOCs. This study shows a novel strategy to exploit the metabolic potential of Actinomycetes as well as emphasizes the importance of studying the interactions between different microorganisms sharing the same ecological niche.

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.

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.

Isothiourea-catalysed enantioselective radical conjugate addition under batch and flow conditions.

The photocatalytic generation of α-amino radicals is combined with chiral isothiourea derived α,ß-unsaturated acyl ammonium intermediates. The reaction proceeds VIA a [3+2] radical-polar crossover mechanism to generate γ-lactams in good yields and enantioselectivities. The enantioselective radical conjugate addition was carried out under batch and flow conditions.

Nickel-Catalysed Cross-Electrophile Coupling of Benzyl Bromides and Sulfonium Salts towards the Synthesis of Dihydrostilbenes.

A nickel-catalysed reductive cross-coupling reaction between benzyl sulfonium salts and benzyl bromides is reported. Simple, stable and readily available sulfonium salts have shown their ability as leaving groups in cross-electrophile coupling, allowing the formation of challenging sp3 -sp3 carbon-carbon bonds, towards the synthesis of interesting dihydrostilbene derivatives. In addition, benzyl tosyl derivatives have been demonstrated to be suitable substrates for reductive cross-coupling by in-situ formation of the corresponding sulfonium salt.

General electrochemical Minisci alkylation of N-heteroarenes with alkyl halides.

Herein, we report, a general, facile and environmentally friendly Minisci-type alkylation of N-heteroarenes under simple and straightforward electrochemical conditions using widely available alkyl halides as radical precursors. Primary, secondary and tertiary alkyl radicals have been shown to be efficiently generated and coupled with a large variety of N-heteroarenes. The method presents a very high functional group tolerance, including various heterocyclic-based natural products, which highlights the robustness of the methodology. This applicability has been further proved in the synthesis of various interesting biologically valuable building blocks. In addition, we have proposed a mechanism based on different proofs and pieces of electrochemical evidence.

Quantification and health risk assessment of lead and cadmium in wheat, rice, and their processed products from Peru.

In Peru, rice grains, wheat, and their processed products are accessible due to their low cost; however, their sale does not have quality certification, so their safety is not guaranteed. This study quantified lead (Pb) and cadmium (Cd) by voltammetry in 16 samples of grains and processed products from four markets in Arequipa (Altiplano, Andrés Avelino Cáceres, Los Incas, and San Camilo) and evaluated their potential health risk. The maximum concentrations of Pb in rice, wheat, and their processed products were 4.821 mg/kg, 7.962 mg/kg, 4.717 mg/kg, and 6.440 mg/kg, respectively; only seven samples showed Cd. All samples exceeded the maximum level (ML) for Pb, and four samples exceeded the ML for Cd established by the Codex Alimentarius (0.200 mg/kg); the rice product had the highest concentration of Pb and Cd. In relation to the estimation of potential health risk, the estimated daily intake (EDI), target hazard quotient (THQ), and target cancer risk (TR), showed that the consumption of all processed rice and wheat products (except Andrés Avelino Cáceres rice and San Camilo wheat) represent a health threat associated with an increased probability of cancer development.

Identification of Antimicrobial Compounds in Two Streptomyces sp. Strains Isolated From Beehives.

The World Health Organization warns that the alarming increase in antibiotic resistant bacteria will lead to 2.7 million deaths annually due to the lack of effective antibiotic therapies. Clearly, there is an urgent need for short-term alternatives that help to alleviate these alarming figures. In this respect, the scientific community is exploring neglected ecological niches from which the prototypical antibiotic-producing bacteria Streptomycetes are expected to be present. Recent studies have reported that honeybees and their products carry Streptomyces species that possess strong antibacterial activity. In this study, we have investigated the antibiotic profile of two Streptomycetes strains that were isolated from beehives. One of the isolates is the strain Streptomyces albus AN1, which derives from pollen, and shows potent antimicrobial activity against Candida albicans. The other isolate is the strain Streptomyces griseoaurantiacus AD2, which was isolated from honey, and displays a broad range of antimicrobial activity against different Gram-positive bacteria, including pathogens such as Staphylococcus aureus and Enterococus faecalis. Cultures of S. griseoaurantiacus AD2 have the capacity to produce the antibacterial compounds undecylprodigiosin and manumycin, while those of S. albus AN1 accumulate antifungal compounds such as candicidins and antimycins. Furthermore, genome and dereplication analyses suggest that the number of putative bioactive metabolites produced by AD2 and AN1 is considerably high, including compounds with anti-microbial and anti-cancer properties. Our results postulate that beehives are a promising source for the discovery of novel bioactive compounds that might be of interest to the agri-food sector and healthcare pharmaceuticals.

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.

A Multidisciplinary Approach to Unraveling the Natural Product Biosynthetic Potential of a Streptomyces Strain Collection Isolated from Leaf-Cutting Ants.

The rapid emergence of bacterial resistance to antibiotics has urged the need to find novel bioactive compounds against resistant microorganisms. For that purpose, different strategies are being followed, one of them being exploring secondary metabolite production in microorganisms from uncommon sources. In this work, we have analyzed the genome of 12 Streptomyces sp. strains of the CS collection isolated from the surface of leaf-cutting ants of the Attini tribe and compared them to four Streptomyces model species and Pseudonocardia sp. Ae150A_Ps1, which shares the ecological niche with those of the CS collection. We used a combination of phylogenetics, bioinformatics and dereplication analysis to study the biosynthetic potential of our strains. 51.5% of the biosynthetic gene clusters (BGCs) predicted by antiSMASH were unknown and over half of them were strain-specific, making this strain collection an interesting source of putative novel compounds.

Enantioselective Inverse-Electron Demand Aza-Diels-Alder Reaction: ipso,α-Selectivity of Silyl Dienol Ethers.

A highly efficient enantioselective inverse-electron-demand aza-Diels-Alder reaction between aza-sulfonyl-1-aza-1,3-butadienes and silyl (di)enol ethers has been developed. The presented methodology allows the synthesis of benzofuran-fused 2-piperidinol derivatives with three contiguous stereocenters in a highly selective manner, as even the hemiaminal center is completely stereocontrolled. Density functional theory (DFT) calculations support that the hydrogen-bond donor-based bifunctional organocatalyst selectively triggers the reaction through the ipso,α-position of the dienophile, in contrast to the reactivity observed for dienolates in situ generated from ß,γ-unsaturated derivatives. Moreover, the calculations have clarified the mechanism of the reaction and the ability of the hydrogen-bond donor core to hydrolyze selectively the E isomer of the dienol ether. Furthermore, to demonstrate the applicability of silyl enol ethers as nucleophiles in the asymmetric synthesis of interesting benzofuran-fused derivatives, the catalytic system has also been implemented for the highly efficient installation of an aromatic ring in the piperidine adducts.

Enantioselective vinylogous Mukaiyama aldol reaction of α-ketoesters under bifunctional organocatalysis.

A highly enantioselective vinylogous Mukaiyama aldol reaction to ketoesters catalysed by a hydrogen-bond-donor-based bifunctional organocatalyst is presented. The addition of silyloxy dienol ether gives rise to multifunctional chiral tertiary alcohols bearing a versatile α,ß-unsaturated aldehyde with excellent enantiocontrol.

Enantioselective vinylogous-Mukaiyama-type dearomatisation by anion-binding catalysis.

The first enantioselective vinylogous Mukaiyama-type dearomatisation of heteroarenes under anion-binding catalysis is presented. A recyclable tetrakistriazole catalyst was used for the enantiocontrol of the remote vinylogous active position of silyl dienol ethers. This approach provided chiral heterocycles bearing α,ß-unsaturated chains with complete regioselectivity and excellent enantioselectivities (up to 97.5 : 2.5 e.r.).