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Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks‐knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection., LaeA and GOX are critical factors modulating A. carbonarius pathogenicity in fruits by controlling transcription of genes important for fungal secondary metabolism and infection; OTA does not contribute to A. carbonarius virulence.

Lichens are prolific producers of natural products of polyketide origin. We previously described a culture of lichen-forming fungus (LFF) Cladonia macilenta that produces biruloquinone, a purple pigment that is a phenanthraquinone rarely found in nature. However, there was no genetic information on the biosynthesis of biruloquinone. To identify a biosynthetic gene cluster for biruloquinone, we mined polyketide synthase (PKS) genes from the genome sequence of a LFF isolated from thalli of C. macilenta. The 38 PKS in C. macilenta are highly diverse, many of which form phylogenetic clades with PKS previously characterized in non-lichenized fungi. We compared transcriptional profiles of the 38 PKS genes in two chemotypic variants, one producing biruloquinone and the other producing no appreciable metabolite in vitro. We identified a PKS gene (hereafter PKS21) that was highly upregulated in the LFF that produces biruloquinone. The boundaries of a putative biruloquinone gene cluster were demarcated by co-expression patterns of six clustered genes, including the PKS21. Biruloquinone gene clusters exhibited a high degree of synteny between related species. In this study we identified a novel PKS family responsible for the biosynthesis of biruloquinone through whole-transcriptome analysis.

Xenorhabdus hominickii ANU1 is known to be an entomopathogenic bacterium symbiotic to nematode Steinernema monticolum. Another bacterial strain X. hominickii DY1 was isolated from a local population of S. monticolum. This bacterial strain X. hominickii DY1 was found to exhibit high insecticidal activities against lepidopteran and coleopteran species after hemocoelic injection. However, these two X. hominickii strains exhibited significant variations in insecticidal activities, with ANU1 strain being more potent than DY1 strain. To clarify their virulence difference, bacterial culture broths of these two strains were compared for secondary metabolite compositions. GC-MS analysis revealed that these two strains had different compositions, including pyrrolopyrazines, piperazines, cyclopeptides, and indoles. Some of these compounds exhibited inhibitory activities against phospholipase A2 to block eicosanoid biosynthesis and induce significant immunosuppression. They also exhibited significant insecticidal activities after oral feeding, with indole derivatives being the most potent. More kinds of indole derivatives were detected in the culture broth of ANU1 strain. To investigate variations in regulation of secondary metabolite production, expression level of leucine-responsive regulatory protein (Lrp), a global transcription factor, was compared. ANU1 strain exhibited significantly lower Lrp expression level than DY1 strain. To assess genetic variations associated with secondary metabolite synthesis, bacterial loci encoding non-ribosomal protein synthase and polyketide synthase (NRPS-PKS) were compared. Three NRPS and four PKS loci were predicted from the genome of X. hominickii. The two bacterial strains exhibited genetic variations (0.12∼0.67%) in amino acid sequences of these NRPS-PKS. Most NRPS-PKS genes exhibited high expression peaks at stationary phase of bacterial growth. However, their expression levels were significantly different between the two strains. These results suggest that differential virulence of the two bacterial strains is caused by the difference in Lrp expression level, leading to difference in the production of indole compounds and other NRPS-PKS-associated secondary metabolites.

Pyrenophora is a fungal genus responsible for a number of major cereal diseases. Although fungi produce many specialised or secondary metabolites for defence and interacting with the surrounding environment, the repertoire of specialised metabolites (SM) within Pyrenophora pathogenic species remains mostly uncharted. In this study, an in-depth comparative analysis of the P. teres f. teres, P teres f. maculata and P. tritici-repentis potential to produce SMs, based on in silico predicted biosynthetic gene clusters (BGCs), was conducted using genome assemblies from PacBio DNA reads. Conservation of BGCs between the Pyrenophora species included type I polyketide synthases, terpene synthases and the first reporting of a type III polyketide synthase in P teres f. maculata. P. teres isolates exhibited substantial expansion of non-ribosomal peptide synthases relative to P. tritici-repentis, hallmarked by the presence of tailoring cis-acting nitrogen methyltransferase domains. P. teres isolates also possessed unique non-ribosomal peptide synthase (NRPS)-indole and indole BGCs, while a P. tritici-repentis phytotoxin BGC for triticone production was absent in P. teres. These differences highlight diversification between the pathogens that reflects their different evolutionary histories, host adaption and lifestyles.

This work presents the identification and proposed biosynthetic pathway for a compound of mixed polyketide-nonribosomal peptide origin that we named acurin A. The compound was isolated from an extract of the filamentous fungus Aspergillus aculeatus, and its core structure resemble that of the mycotoxin fusarin C produced by several Fusarium species. Based on bioinformatics in combination with RT-qPCR experiments and gene-deletion analysis, we identified a biosynthetic gene cluster (BGC) in A. aculeatus responsible for the biosynthesis of acurin A. Moreover, we were able to show that a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) enzyme separately encoded by this BGC are responsible for the synthesis of the PK-NRP compound, acurin A, core structure. In comparison, the production of fusarin C is reported to be facilitated by a linked PKS-NRPS hybrid enzyme. Phylogenetic analyses suggest the PKS and NRPS in A. aculeatus resulted from a recent fission of an ancestral hybrid enzyme followed by gene duplication. In addition to the PKS- and NRPS-encoding genes of acurin A, we show that six other genes are influencing the biosynthesis including a regulatory transcription factor. Altogether, we have demonstrated the involvement of eight genes in the biosynthesis of acurin A, including an in-cluster transcription factor. This study highlights the biosynthetic capacity of A. aculeatus and serves as an example of how the CRISPR/Cas9 system can be exploited for the construction of fungal strains that can be readily engineered.

© 2017 Metabolic engineering efforts toward rewiring metabolism of cells to produce new compounds often require the utilization of non-native enzymatic machinery that is capable of producing a broad range of chemical functionalities. Polyketides encompass one of the largest classes of chemically diverse natural products. With thousands of known polyketides, modular polyketide synthases (PKSs) share a particularly attractive biosynthetic logic for generating chemical diversity. The engineering of modular PKSs could open access to the deliberate production of both existing and novel compounds. In this review, we discuss PKS engineering efforts applied at both the protein and cellular level for the generation of a diverse range of chemical structures, and we examine future applications of PKSs in the production of medicines, fuels and other industrially relevant chemicals.

The prevalence of antibiotic resistance and the decrease in novel antibiotic discovery in recent years necessitates the identification of potentially novel microbial resources to produce natural products. Ktedonobacteria, a class of deeply branched bacterial lineage in the ancient phylum Chloroflexi, are ubiquitous in terrestrial environments and characterized by their large genome size and complex life cycle. These characteristics indicate Ktedonobacteria as a potential active producer of bioactive compounds. In this study, we observed the existence of a putative "megaplasmid," multiple copies of ribosomal RNA operons, and high ratio of hypothetical proteins with unknown functions in the class Ktedonobacteria. Furthermore, a total of 104 antiSMASH-predicted putative biosynthetic gene clusters (BGCs) for secondary metabolites with high novelty and diversity were identified in nine Ktedonobacteria genomes. Our investigation of domain composition and organization of the non-ribosomal peptide synthetase and polyketide synthase BGCs further supports the concept that class Ktedonobacteria may produce compounds structurally different from known natural products. Furthermore, screening of bioactive compounds from representative Ktedonobacteria strains resulted in the identification of broad antimicrobial activities against both Gram-positive and Gram-negative tested bacterial strains. Based on these findings, we propose the ancient, ubiquitous, and spore-forming Ktedonobacteria as a versatile and promising microbial resource for natural product discovery.

There is an urgent need to search for new antibiotics to counter the growing number of antibiotic-resistant bacterial strains, one of which is methicillin-resistant Staphylococcus aureus (MRSA). Herein, we report a Streptomyces sp. strain MUSC 125 from mangrove soil in Malaysia which was identified using 16S rRNA phylogenetic and phenotypic analysis. The methanolic extract of strain MUSC 125 showed anti-MRSA, anti-biofilm and antioxidant activities. Strain MUSC 125 was further screened for the presence of secondary metabolite biosynthetic genes. Our results indicated that both polyketide synthase (pks) gene clusters, pksI and pksII, were detected in strain MUSC 125 by PCR amplification. In addition, gas chromatography-mass spectroscopy (GC-MS) detected the presence of different chemicals in the methanolic extract. Based on the GC-MS analysis, eight known compounds were detected suggesting their contribution towards the anti-MRSA and anti-biofilm activities observed. Overall, the study bolsters the potential of strain MUSC 125 as a promising source of anti-MRSA and antibiofilm compounds and warrants further investigation.

Marine environments harbor a wide range of microorganisms from the three domains of life. These microorganisms have great potential to enable discovery of new enzymes and bioactive compounds for industrial use. However, only ~1% of microorganisms from the environment can currently be identified through cultured isolates, limiting the discovery of new compounds. To overcome this limitation, a metagenomics approach has been widely adopted for biodiversity studies on samples from marine environments. In this study, we screened metagenomes in order to estimate the potential for new natural compound synthesis mediated by diversity in the Polyketide Synthase (PKS) and Nonribosomal Peptide Synthetase (NRPS) genes. The samples were collected from the Praia dos Anjos (Angel's Beach) surface water-Arraial do Cabo (Rio de Janeiro state, Brazil), an environment affected by upwelling. In order to evaluate the potential for screening natural products in Arraial do Cabo samples, we used KS (keto-synthase) and C (condensation) domains (from PKS and NRPS, respectively) to build Hidden Markov Models (HMM) models. From both samples, a total of 84 KS and 46 C novel domain sequences were obtained, showing the potential of this environment for the discovery of new genes of biotechnological interest. These domains were classified by phylogenetic analysis and this was the first study conducted to screen PKS and NRPS genes in an upwelling affected sample.

Department of Chemistry, Nanoscience Center, University of Jyväskylä, FI-40014, Jyväskylä, Finland Cyclodextrins are cyclic oligosaccharides widely used in the pharmaceutical industry to improve drug delivery and to increase the solubility of hydrophobic compounds. Anabaenolysins are lipopeptides produced by cyanobacteria with potent lytic activity in cholesterolcontaining membranes. Here, we identified the 23- To 24-kb gene clusters responsible for the production of the lipopeptide anabaenolysin. The hybrid nonribosomal peptide synthetase and polyketide synthase biosynthetic gene cluster is encoded in the genomes of three anabaenolysin-producing strains of Anabaena.We detected previously unidentified strains producing known anabaenolysins A and B and discovered the production of new variants of anabaenolysins C and D. Bioassays demonstrated that anabaenolysins have weak antifungal activity against Candida albicans. Surprisingly, addition of the hydrophilic fraction of the whole-cell extracts increased the antifungal activity of the hydrophobic anabaenolysins. The fraction contained compounds identified by NMR as ?-, ?-, and ?-cyclodextrins, which undergo acetylation. Cyclodextrins have been used for decades to improve the solubility and bioavailability of many drugs including antifungal compounds. This study shows a natural example of cyclodextrins improving the solubility and efficacy of an antifungal compound in an ancient lineage of photosynthetic bacteria.

A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an "assembly line" and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such "unnatural" natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)--discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts.

Fusarielins are polyketides with a decalin core produced by various species of Aspergillus and Fusarium. Although the responsible gene cluster has been identified, the biosynthetic pathway remains to be elucidated. In the present study, members of the gene cluster were deleted individually in a Fusarium graminearum strain overexpressing the local transcription factor. The results suggest that a trans-acting enoyl reductase (FSL5) assists the polyketide synthase FSL1 in biosynthesis of a polyketide product, which is released by hydrolysis by a trans-acting thioesterase (FSL2). Deletion of the epimerase (FSL3) resulted in accumulation of an unstable compound, which could be the released product. A novel compound, named prefusarielin, accumulated in the deletion mutant of the cytochrome P450 monooxygenase FSL4. Unlike the known fusarielins from Fusarium, this compound does not contain oxygenized decalin rings, suggesting that FSL4 is responsible for the oxygenation.

Cyanobacteria are prolific producers of natural products. Investigations into the biochemistry responsible for the formation of these compounds have revealed fascinating mechanisms that are not, or only rarely, found in other microorganisms. In this article, we survey the biosynthetic pathways of cyanobacteria isolated from freshwater, marine and terrestrial habitats. We especially emphasize modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) pathways and highlight the unique enzyme mechanisms that were elucidated or can be anticipated for the individual products. We further include ribosomal natural products and UV-absorbing pigments from cyanobacteria. Mechanistic insights obtained from the biochemical studies of cyanobacterial pathways can inspire the development of concepts for the design of bioactive compounds by synthetic-biology approaches in the future.

Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes.

Non-ribosomal peptide synthesis in fungi requires a ready supply of proteogenic and non-proteogenic amino acids which are subsequently incorporated into the nascent non-ribosomal peptide via a thiotemplate mechanism catalysed by non-ribosomal peptide synthetases. Substrate amino acids can be modified prior to or during incorporation into the non-ribosomal peptide, or following incorporation into an early stage amino acid-containing biosynthetic intermediate. These post-incorporation modifications involve a range of additional enzymatic activities including but not exclusively, monooxygenases, methyltransferases, epimerases, oxidoreductases and glutathione transferases which are essential to effect biosynthesis of the final non-ribosomal peptide. Likewise, polyketide biosynthesis is directly by polyketide synthase megaenzymes and cluster-encoded ancilliary decorating enzymes. Additionally, a suite of additional primary metabolites, for example: CoA, acetyl CoA, S-adenosylmethionine, glutathione, NADPH, malonyl CoA and molecular oxygen, amongst others are required for non-ribosomal peptide and polyketide synthesis. Clearly these processes must involve exquisite orchestration to facilitate the simultaneous biosynthesis of different types of non-ribosomal peptides, polyketides, and related metabolites requiring identical or similar biosynthetic precursors or co-factors. Moreover, the near identical structures of many natural products within a given family (e.g., ergot alkaloids), along with localization to similar regions within fungi (e.g., conidia) suggests that cross-talk may exist, in terms of biosynthesis and functionality. Finally, we speculate if certain biosynthetic steps involved in non-ribosomal peptide and polyketide synthesis play a role in cellular protection or environmental adaptation, and wonder if these enzymatic reactions are of equivalent importance to the actual biosynthesis of the final metabolite.

Background Cyanobacteria are an ancient lineage of photosynthetic bacteria from which hundreds of natural products have been described, including many notorious toxins but also potent natural products of interest to the pharmaceutical and biotechnological industries. Many of these compounds are the products of non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways. However, current understanding of the diversification of these pathways is largely based on the chemical structure of the bioactive compounds, while the evolutionary forces driving their remarkable chemical diversity are poorly understood. Results We carried out a phylum-wide investigation of genetic diversification of the cyanobacterial NRPS and PKS pathways for the production of bioactive compounds. 452 NRPS and PKS gene clusters were identified from 89 cyanobacterial genomes, revealing a clear burst in late-branching lineages. Our genomic analysis further grouped the clusters into 286 highly diversified cluster families (CF) of pathways. Some CFs appeared vertically inherited, while others presented a more complex evolutionary history. Only a few horizontal gene transfers were evidenced amongst strongly conserved CFs in the phylum, while several others have undergone drastic gene shuffling events, which could result in the observed diversification of the pathways. Conclusions Therefore, in addition to toxin production, several NRPS and PKS gene clusters are devoted to important cellular processes of these bacteria such as nitrogen fixation and iron uptake. The majority of the biosynthetic clusters identified here have unknown end products, highlighting the power of genome mining for the discovery of new natural products. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-977) contains supplementary material, which is available to authorized users.

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA comprises a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described in A. carbonarius, which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the beta-ketosynthase and acyl-transferase domains of the OTA PKSs that had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A quantitative RT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed, with AcOTApks reaching its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production. (C) 2014 Elsevier B.V. All rights reserved.

Sponge-associated microorganisms are able to assemble the complex machinery for the production of secondary metabolites such as polyketides, the most important class of marine natural products from a drug discovery perspective. A comprehensive overview of polyketide biosynthetic genes of the sponge Plakortis halichondrioides and its symbionts was obtained in the present study by massively parallel 454 pyrosequencing of complex and heterogeneous PCR (Polymerase Chain Reaction) products amplified from the metagenomic DNA of a specimen of P. halichondrioides collected in the Caribbean Sea. This was accompanied by a survey of the bacterial diversity within the sponge. In line with previous studies, sequences belonging to supA and swfA, two widespread sponge-specific groups of polyketide synthase (PKS) genes were dominant. While they have been previously reported as belonging to Poribacteria (a novel bacterial phylum found exclusively in sponges), re-examination of current genomic sequencing data showed supA and swfA not to be present in the poribacterial genome. Several non-supA, non-swfA type-I PKS fragments were also identified. A significant portion of these fragments resembled type-I PKSs from protists, suggesting that bacteria may not be the only source of polyketides from P. halichondrioides, and that protistan PKSs should receive further investigation as a source of novel polyketides.

Cladosporium cladosporioides is a dematiaceous fungus with coloured mycelia and conidia due to the presence of dark pigments. The purpose of this study was to characterize the dark pigments synthetized by Cladosporium sp. LPSC no. 1088 and also to identify the putative polyketide synthase (pks) gene that might be involved in the pigment biosynthesis. Morphological as well as molecular features like the ITS sequence confirmed that LPSC 1088 is Cladosporium cladosporioides. UV-visible, Fourier Transform Infrared (FTIR) and Electron Spin Resonance (ESR) spectroscopy analysis as well as melanin inhibitors suggest that the main dark pigment of the isolate was 1,8 dihydroxynaphthalene (DHN)-melanin-type compound. Two commercial fungicides, Difenoconazole and Chlorothalonil, inhibited fungal growth as well as increased pigmentation of the colonies suggesting that melanin might protect the fungus against chemical stress. The pigment is most probably synthetized by means of a pentaketide pathway since the sequence of a 651 bp fragment, coding for a putative polyketide synthase, is highly homologous to pks sequences from other fungi. Fil: Llorente, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bárcena, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vera Bahima, José. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Saparrat, Mario Carlos Nazareno. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Botánica Spegazzini; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Arambarri, Angélica Margarita. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Botánica Spegazzini; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rozas, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mirifico, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Balatti, Pedro Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales; Argentina

International audience; The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquidchromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminalACP domain of Pks13 (N-ACPPks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACPPks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce -alkyl -ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterialdrug screening.

Myxobacteria belonging to the genus Sorangium are known to produce a variety of biologically active secondary metabolites. Chivosazol is a macrocyclic antibiotic active against yeast, filamentous fungi and especially against mammalian cells. The compound specifically destroys the actin skeleton of eucaryotic cells and does not show activity against bacteria. Chivosazol contains an oxazole ring and a glycosidically bound 6-deoxyglucose (except for chivosazol F). In this paper we describe the biosynthetic gene cluster that directs chivosazol biosynthesis in the model strain Sorangium cellulosum So ce56. This biosynthetic gene cluster spans 92 kbp on the chromosome and contains four polyketide synthase genes and one hybrid polyketide synthase/nonribosomal peptide synthetase gene. An additional gene encoding a protein with similarity to different methyltransferases and presumably involved in post-polyketide modification was identified downstream of the core biosynthetic gene cluster. The chivosazol biosynthetic gene locus belongs to the recently identified and rapidly growing class of trans-acyltransferase polyketide synthases, which do not contain acyltransferase domains integrated into the multimodular megasynthetases. (c) 2005 Elsevier B.V. All rights reserved.