Volatile communication in Actinobacteria: a language for secondary metabolism regulation.

BACKGROUND: Volatile compounds are key elements in the interaction and communication between organisms at both interspecific and intraspecific levels. In complex bacterial communities, the emission of these fast-acting chemical messengers allows an exchange of information even at a certain distance that can cause different types of responses in the receiving organisms. The changes in secondary metabolism as a consequence of this interaction arouse great interest in the field of searching for bioactive compounds since they can be used as a tool to activate silenced metabolic pathways. Regarding the great metabolic potential that the Actinobacteria group presents in the production of compounds with attractive properties, we evaluated the reply the emitted volatile compounds can generate in other individuals of the same group. RESULTS: We recently reported that volatile compounds released by different streptomycete species trigger the modulation of biosynthetic gene clusters in Streptomyces spp. which finally leads to the activation/repression of the production of secondary metabolites in the recipient strains. Here we present the application of this rationale in a broader bacterial community to evaluate volatiles as signaling effectors that drive the activation of biosynthesis of bioactive compounds in other members of the Actinobacteria group. Using cocultures of different actinobacteria (where only the volatile compounds reach the recipient strain) we were able to modify the bacterial secondary metabolism that drives overproduction (e.g., granaticins, actiphenol, chromomycins) and/or de novo production (e.g., collismycins, skyllamycins, cosmomycins) of compounds belonging to different chemical species that present important biological activities. CONCLUSIONS: This work shows how the secondary metabolism of different Actinobacteria species can vary significantly when exposed in co-culture to the volatile compounds of other phylum-shared bacteria, these effects being variable depending on strains and culture media. This approach can be applied to the field of new drug discovery to increase the battery of bioactive compounds produced by bacteria that can potentially be used in treatments for humans and animals.

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.

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.

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.

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.

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.

Organocatalytic Strategies for the Development of the Enantioselective Inverse-electron-demand Hetero-Diels-Alder Reaction.

Cycloaddition reactions, in particular Diels-Alder reactions, have attracted a lot of attention from organic chemists since they represent one of the most powerful methodologies for the construction of carbon-carbon bonds. In particular, inverse-electron-demand hetero-Diels-Alder reactions have been an important breakthrough for the synthesis of heterocyclic compounds. Among all their variants, the organocatalytic enantioselective version has been widely explored since the asymmetric construction of diversely functionalized scaffolds under reaction conditions encompassed within the green chemistry field is of great interest. In this review, a profound revision on the latest advances on the organocatalytic asymmetric inverse-electron demand hetero-Diels-Alder reaction is shown.

New Sipanmycin Analogues Generated by Combinatorial Biosynthesis and Mutasynthesis Approaches Relying on the Substrate Flexibility of Key Enzymes in the Biosynthetic Pathway.

The appearance of new infectious diseases, the increase in multidrug-resistant bacteria, and the need for more effective chemotherapeutic agents have oriented the interests of researchers toward the search for metabolites with novel or improved bioactivities. Sipanmycins are disaccharyl glycosylated macrolactams that exert antibiotic and cytotoxic activities. By applying combinatorial biosynthesis and mutasynthesis approaches, we have generated eight new members of the sipanmycin family. The introduction of plasmids harboring genes responsible for the biosynthesis of several deoxysugars into sipanmycin-producing Streptomyces sp. strain CS149 led to the production of six derivatives with altered glycosylation patterns. After structural elucidation of these new metabolites, we conclude that some of these sugars are the result of the combination of the enzymatic machinery encoded by the introduced plasmids and the native enzymes of the d-sipanose biosynthetic pathway of the wild-type CS149 strain. In addition, two analogues of the parental compounds with a modified polyketide backbone were generated by a mutasynthesis approach, feeding cultures of a mutant strain defective in sipanmycin biosynthesis with 3-aminopentanoic acid. The generation of new sipanmycin analogues shown in this work relied on the substrate flexibility of key enzymes involved in sipanmycin biosynthesis, particularly the glycosyltransferase pair SipS9/SipS14 and enzymes SipL3, SipL1, SipL7, and SipL2, which are involved in the incorporation of the polyketide synthase starting unit.IMPORTANCE Combinatorial biosynthesis has proved its usefulness in generating derivatives of already known compounds with novel or improved pharmacological properties. Sipanmycins are a family of glycosylated macrolactams produced by Streptomyces sp. strain CS149, whose antiproliferative activity is dependent on the sugar moieties attached to the aglycone. In this work, we report the generation of several sipanmycin analogues with different deoxysugars, showing the high degree of flexibility exerted by the glycosyltransferase machinery with respect to the recognition of diverse nucleotide-activated sugars. In addition, modifications in the macrolactam ring were introduced by mutasynthesis approaches, indicating that the enzymes involved in incorporating the starter unit have a moderate ability to introduce different types of ß-amino acids. In conclusion, we have proved the substrate flexibility of key enzymes involved in sipanmycin biosynthesis, especially the glycosyltransferases, which can be exploited in future experiments.

Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin.

BACKGROUND: Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of mithramycin proceeds through the formation of the key intermediate 4-demethyl-premithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to mithramycin. However, the biosynthesis pathway for 4-demethyl-premithramycinone remains unclear. RESULTS: Expression of cosmid cosAR7, containing a set of mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-premithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-premithramycinone. Expression of different sets of mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, assigning gene functions and establishing the order of biosynthetic steps. CONCLUSIONS: We established the biosynthesis pathway for 4-demethyl-premithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7-C12), followed by formation of the second and third rings (C5-C14; C3-C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1-C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-premithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new mithramycin analogs and improve their production titers for bioactivity investigation.

Cooperative Involvement of Glycosyltransferases in the Transfer of Amino Sugars during the Biosynthesis of the Macrolactam Sipanmycin by Streptomyces sp. Strain CS149.

Macrolactams comprise a family of natural compounds with important bioactivities, such as antibiotic, antifungal, and antiproliferative activities. Sipanmycins A and B are two novel members of this family, with two sugar moieties attached to the aglycon. In the related macrolactam vicenistatin, the sugar moiety has been proven to be essential for cytotoxicity. In this work, the gene cluster responsible for the biosynthesis of sipanmycins (sip cluster) in Streptomyces sp. strain CS149 is described and the steps involved in the glycosylation of the final compounds unraveled. Also, the cooperation of two different glycosyltransferases in each glycosylation step is demonstrated. Additionally, the essential role of SipO2 as an auxiliary protein in the incorporation of the second deoxy sugar is addressed. In light of the results obtained by the generation of mutant strains and in silico characterization of the sip cluster, a biosynthetic pathway for sipanmycins and the two deoxy sugars attached is proposed. Finally, the importance of the hydroxyl group at C-10 of the macrolactam ring and the sugar moieties for cytotoxicity and antibiotic activity of sipanmycins is shown.IMPORTANCE The rapid emergence of infectious diseases and multiresistant pathogens has increased the necessity for new bioactive compounds; thus, novel strategies have to be developed to find them. Actinomycetes isolated in symbiosis with insects have attracted attention in recent years as producers of metabolites with important bioactivities. Sipanmycins are glycosylated macrolactams produced by Streptomyces sp. CS149, isolated from leaf-cutting ants, and show potent cytotoxic activity. Here, we characterize the sip cluster and propose a biosynthetic pathway for sipanmycins. As far as we know, it is the first time that the cooperation between two different glycosyltransferases is demonstrated to be strictly necessary for the incorporation of the same sugar. Also, a third protein with homology to P450 monooxygenases, SipO2, is shown to be essential in the second glycosylation step, forming a complex with the glycosyltransferase pair SipS9-SipS14.

Reformatsky Reaction to Alkynyl N- tert-Butanesulfinyl Imines: Lewis Acid Controlled Stereodivergent Synthesis of ß-Alkynyl-ß-Amino Acids.

A highly diastereoselective Refortmatsky reaction to N- tert-butanesulfinyl propargylaldimines and ketimines is presented. The reaction proceeded with excellent yields and diastereoselectivities provided by the sulfinyl group in the presence of Me3Al. The use of TBSOTf as a Lewis acid promoter switched the sense of the stereoinduction. Thus, this methodology allowed the stereodivergent asymmetric synthesis of ß-alkynyl ß-amino acid derivatives, from the same sulfinyl configuration, by simply changing the Lewis acid promoter.

Characterization of the Jomthonic Acids Biosynthesis Pathway and Isolation of Novel Analogues in Streptomyces caniferus GUA-06-05-006A.

Jomthonic acids (JAs) are a group of natural products (NPs) with adipogenic activity. Structurally, JAs are formed by a modified ß-methylphenylalanine residue, whose biosynthesis involves a methyltransferase that in Streptomyces hygroscopicus has been identified as MppJ. Up to date, three JA members (A⁻C) and a few other natural products containing ß-methylphenylalanine have been discovered from soil-derived microorganisms. Herein, we report the identification of a gene (jomM) coding for a putative methyltransferase highly identical to MppJ in the chromosome of the marine actinobacteria Streptomyces caniferus GUA-06-05-006A. In its 5' region, jomM clusters with two polyketide synthases (PKS) (jomP1, jomP2), a nonribosomal peptide synthetase (NRPS) (jomN) and a thioesterase gene (jomT), possibly conforming a single transcriptional unit. Insertion of a strong constitutive promoter upstream of jomP1 led to the detection of JA A, along with at least two novel JA family members (D and E). Independent inactivation of jomP1, jomN and jomM abolished production of JA A, JA D and JA E, indicating the involvement of these genes in JA biosynthesis. Heterologous expression of the JA biosynthesis cluster in Streptomyces coelicolor M1152 and in Streptomyces albus J1074 led to the production of JA A, B, C and F. We propose a pathway for JAs biosynthesis based on the findings here described.

An Interrupted Pummerer/Nickel-Catalysed Cross-Coupling Sequence.

An interrupted Pummerer/nickel-catalysed cross-coupling strategy has been developed and used in the elaboration of styrenes. The operationally simple method can be carried out as a one-pot process, involves the direct formation of stable alkenyl sulfonium salt intermediates, utilises a commercially available sulfoxide, catalyst, and ligand, operates at ambient temperature, accommodates sp-, sp2 -, and sp3 -hybridised organozinc coupling partners, and delivers functionalised styrene products in high yields over two steps. An interrupted Pummerer/cyclisation approach has also been used to access carbo- and heterocyclic alkenyl sulfonium salts for cross-coupling.

Synthesis of C2 Substituted Benzothiophenes via an Interrupted Pummerer/[3,3]-Sigmatropic/1,2-Migration Cascade of Benzothiophene S-Oxides.

Functionalized benzothiophenes are important scaffolds found in molecules with wide ranging biological activity and in organic materials. We describe an efficient, metal-free synthesis of C2 arylated, allylated, and propargylated benzothiophenes. The reaction utilizes synthetically unexplored yet readily accessible benzothiophene S-oxides and phenols, allyl-, or propargyl silanes in a unique cascade sequence. An interrupted Pummerer reaction between benzothiophene S-oxides and the coupling partners yields sulfonium salts that lack aromaticity and therefore allow facile [3,3]-sigmatropic rearrangement. The subsequently generated benzothiophenium salts undergo a previously unexplored 1,2-migration to access C2 functionalized benzothiophenes.

Exploring the biocombinatorial potential of benzoxazoles: generation of novel caboxamycin derivatives.

BACKGROUND: The biosynthesis pathway of benzoxazole compounds caboxamycin and nataxazole have been recently elucidated. Both compounds share one of their precursors, 3-hydroxyanthranilate (two units in the case of nataxazole). In addition, caboxamycin structure includes a salicylate moiety while 6-methylsalycilate is the third scaffold in nataxazole. Pathways cross-talk has been identified in caboxamycin producer Streptomyces sp. NTK937, between caboxamycin and enterobactin pathways, and nataxazole producer Streptomyces sp. Tü6176, between nataxazole and coelibactin pathways. These events represent a natural form of combinatorial biosynthesis. RESULTS: Eleven novel caboxamycin derivatives, and five putative novel derivatives, bearing distinct substitutions in the aryl ring have been generated. These compounds were produced by heterologous expression of several caboxamycin biosynthesis genes in Streptomyces albus J1074 (two compounds), by combinatorial biosynthesis in Streptomyces sp. NTK937 expressing nataxazole iterative polyketide synthase (two compounds) and by mutasynthesis using a nonproducing mutant of Streptomyces sp. NTK937 (12 compounds). Some of the compounds showed improved bioactive properties in comparison with caboxamycin. CONCLUSIONS: In addition to the benzoxazoles naturally biosynthesized by the caboxamycin and nataxazole producers, a greater structural diversity can be generated by mutasynthesis and heterologous expression of benzoxazole biosynthesis genes, not only in the respective producer strains but also in non-benzoxazole producers such as S. albus strains. These results show that the production of a wide variety of benzoxazoles could be potentially achieved by the sole expression of cbxBCDE genes (or orthologs thereof), supplying an external source of salicylate-like compounds, or with the concomitant expression of other genes capable of synthesizing salicylates, such as cbxA or natPK.

Engineered jadomycin analogues with altered sugar moieties revealing JadS as a substrate flexible O-glycosyltransferase.

Glycosyltransferases (GTs)-mediated glycodiversification studies have drawn significant attention recently, with the goal of generating bioactive compounds with improved pharmacological properties by diversifying the appended sugars. The key to achieving glycodiversification is to identify natural and/or engineered flexible GTs capable of acting upon a broad range of substrates. Here, we report the use of a combinatorial biosynthetic approach to probe the substrate flexibility of JadS, the GT in jadomycin biosynthesis, towards different non-native NDP-sugar substrates, enabling us to identify six jadomycin B analogues with different sugar moieties. Further structural engineering by precursor-directed biosynthesis allowed us to obtain 11 new jadomycin analogues. Our results for the first time show that JadS is a flexible O-GT that can utilize both L- and D- sugars as donor substrates, and tolerate structural changes at the C2, C4 and C6 positions of the sugar moiety. JadS may be further exploited to generate novel glycosylated jadomycin molecules in future glycodiversification studies.

Novel Bioactive Paulomycin Derivatives Produced by Streptomyces albus J1074.

Four novel paulomycin derivatives have been isolated from S. albus J1074 grown in MFE culture medium. These compounds are structural analogs of antibiotics 273a2α and 273a2ß containing a thiazole moiety, probably originated through an intramolecular Michael addition. The novel, thiazole, moiety-containing paulomycins show a lower antibiotic activity than paulomycins A and B against Gram-positive bacteria. However, two of them show an improved activity against Gram-negative bacteria. In addition, the four novel compounds are more stable in culture than paulomycins A and B. Thus, the presence of an N-acetyl-l-cysteine moiety linked to the carbon atom of the paulic acid isothiocyanate moiety, via a thioester bond, and the subsequent intramolecular cyclization of the paulic acid to generate a thiazole heterocycle confer to paulomycins a higher structural stability that otherwise will conduce to paulomycin degradation and into inactive paulomenols.

Metal-Free CH-CH-Type Cross-Coupling of Arenes and Alkynes Directed by a Multifunctional Sulfoxide Group.

A metal-free CH-CH-type coupling of arenes and alkynes, mediated by a multifunctional sulfoxide directing group, exploits nonprefunctionalized coupling partners, proceeds under mild conditions, is operationally simple, and exhibits high functional group tolerance. The products of the CH-CH coupling are highly versatile, and the metal-free process can be used for the construction and late-stage modification of important molecular scaffolds.

New insights into paulomycin biosynthesis pathway in Streptomyces albus J1074 and generation of novel derivatives by combinatorial biosynthesis.

BACKGROUND: Streptomyces albus J1074 produces glycosylated antibiotics paulomycin A, B and E that derive from chorismate and contain an isothiocyanate residue in form of paulic acid. Paulomycins biosynthesis pathway involves two glycosyltransferases, three acyltransferases, enzymes required for paulic acid biosynthesis (in particular an aminotransferase and a sulfotransferase), and enzymes involved in the biosynthesis of two deoxysugar moieties: D-allose and L-paulomycose. RESULTS: Inactivation of genes encoding enzymes involved in deoxysugar biosynthesis, paulic acid biosynthesis, deoxysugar transfer, and acyl moieties transfer has allowed the identification of several biosynthetic intermediates and shunt products, derived from paulomycin intermediates, and to propose a refined version of the paulomycin biosynthesis pathway. Furthermore, several novel bioactive derivatives of paulomycins carrying modifications in the L-paulomycose moiety have been generated by combinatorial biosynthesis using different plasmids that direct the biosynthesis of alternative deoxyhexoses. CONCLUSIONS: The paulomycins biosynthesis pathway has been defined by inactivation of genes encoding glycosyltransferases, acyltransferases and enzymes involved in paulic acid and L-paulomycose biosynthesis. These experiments have allowed the assignment of each of these genes to specific paulomycin biosynthesis steps based on characterization of products accumulated by the corresponding mutant strains. In addition, novel derivatives of paulomycin A and B containing L-paulomycose modified moieties were generated by combinatorial biosynthesis. The production of such derivatives shows that L-paulomycosyl glycosyltransferase Plm12 possesses a certain degree of flexibility for the transfer of different deoxysugars. In addition, the pyruvate dehydrogenase system form by Plm8 and Plm9 is also flexible to catalyze the attachment of a two-carbon side chain, derived from pyruvate, into both 2,6-dideoxyhexoses and 2,3,6-trideoxyhexoses. The activity of the novel paulomycin derivatives carrying modifications in the L-paulomycose moiety is lower than the original compounds pointing to some interesting structure-activity relationships.