Hybrid Polyketide Synthase-nonribosomal Peptide Synthetase Research Articles

Human-associated bacteria adopt an unusual route for synthesizing 3-acetylated tetramates for environmental adaptation

BackgroundTetramates or tetramic acid-containing compounds (TACs) are a group of bioactive natural products featuring a pyrrolidine-2,4-dione ring acknowledged being closed via Dieckmann cyclization. The cariogenic Streptococcus mutans strains bearing a muc biosynthetic gene cluster (BGC) can synthesize mutanocyclin (MUC), a 3-acetylated TAC that can inhibit both leukocyte chemotaxis and filamentous development in Candida albicans. Some strains can also accumulate reutericyclins (RTCs), the intermediates of MUC biosynthesis with antibacterial activities. However, the formation mechanism of the pyrrolidine-2,4-dione ring of MUC and the distribution of muc-like BGCs along with their ecological functions has not been explored extensively.ResultsWe demonstrated that a key intermediate of MUC biosynthesis, M-307, is installed by a hybrid nonribosomal peptide synthetase-polyketide synthase assembly line and its pyrrolidine-2,4-dione ring is closed via an unprecedented lactam bond formation style. Subsequent C-3 acetylation will convert M-307 to RTCs, which is then hydrolyzed by a deacylase, MucF, to remove the N-1 fatty acyl appendage to generate MUC. Distribution analysis showed that the muc-like BGCs distribute predominantly in human-associated bacteria. Interestingly, most of the muc-like BGCs possessing a mucF gene were isolated from human or livestock directly, indicating their involvement in alleviating the host’s immune attacks by synthesizing MUC; while those BGCs lacking mucF gene distribute mainly in bacteria from fermented products, suggesting that they tend to synthesize RTCs to compete with neighboring bacteria. It is noteworthy that many bacteria in the same habitats (e.g., the oral cavity) lack the muc-like BGC, but possess functional MucF homologues to “detoxify” RTCs to MUC, including several competitive bacteria of S. mutans. We also comparably studied the distribution of TAS1, a fungal enzyme responsible for the production of phytotoxic tenuazonic acids (TeAs), a class of 3-acetylated TACs with similar structure but distinct biosynthetic mechanism to MUC, and found that it mainly exists in plants or crops.ConclusionsThe in vivo and in vitro experiments revealed that the pyrrolidine-2,4-dione ring of MUC is closed via lactam bond formation, which may be adopted by many TACs without 3-acyl decorations. Besides, we found that muc-like BGCs are widespread in human-associated bacteria and their shapes and main products can be influenced by the habitat environment and vice versa. By comparing with TeAs, we provided thought-provoking insights into how ecological and evolutionary forces drive bacteria and fungi to construct a common 3-acetylated pyrrolidine-2,4-dione core through different routes, and how the biosynthetic processes are delicately controlled to generate diverse 3-acetylated TACs for environmental adaptation.2PMNhDpkP3itX1xdtgrn3CVideo

Somalactams A-D: Anti-inflammatory Macrolide Lactams with Unique Ring Systems from an Arctic Actinomycete Strain.

Four new PKS-NRPS-derived macrolide lactams with three unique ring fusion types were discovered from the Arctic sponge associated actinomycete Streptomyces somaliensis 1107 using a genome mining strategy. Their structures were elucidated by a combination of MS, NMR spectroscopic analysis, and single-crystal X-ray diffraction. Biosynthetically, a novel gene cluster sml consisting of three polyketide synthases and one hybrid polyketide synthase-nonribosomal peptide synthetase together with cytochrome P450s and flavin-containing monooxygenases and oxidoreductases was demonstrated to assemble the unique skeleton. Pharmacological studies revealed that compound 1 displayed a potent anti-inflammatory effect without cytotoxicity. It inhibited IL-6 and TNF-α release in the serum of LPS-stimulated RAW264.7 macrophage cells with IC50 values of 5.76 and 0.18 μM, respectively, and modulated the MAPK pathway. Moreover, compound 1 alleviated LPS-induced systemic inflammation in our transgenic fluorescent zebrafish model.

Genetic responses to adding nitrates to improve hydrophilic yellow pigment in Monascus fermentation.

Nitrates can stimulate the biosynthesis of hydrophilic yellow pigments (HYPs) in Monascus ruber CGMCC 10910. To explore the molecular mechanisms whereby nitrates (NaNO3 and NH4NO3) regulate HYP production, an integrated transcriptomic and proteomic analysis was conducted in this study. Nitrate addition led to an approximately 75% higher HYP production compared with the untreated group, especially compounds Y3 and Y4. Comparative transcriptomic analysis found that mpigsA, H, K, L, and P genes involved in yellow pigment biosynthesis were significantly upregulated. In addition, pigment biosynthesis-related (carbon catabolism, amino acid metabolism, polyketide synthesis, and fatty acid metabolism) genes were upregulated to provide precursors and energy for HYP biosynthesis and cell growth. Secretion-related (cytomembrane ergosterol biosynthetic, and transport) pathways were also noticeably regulated to accelerate transmembrane transport of HYPs. Meanwhile, proteomicanalysis showed that nitrates improved the protein expression of hybrid polyketide synthase-nonribosomal peptide synthetase, oxidoreductase, glucoamylase, endo-1,4-beta-xylanase, O-acetylhomoserine, and isocitrate lyase to enhance HYP production. These findings demonstrated the regulatory mechanism of nitrates for enhancing HYP production in Monascus. KEY POINTS: • Nitrates stimulated the biosynthesis of Monascus hydrophilic yellow pigments (HYPs) • Nitrates affected transcriptional level of pigment biosynthesis- and transport genes • Increased expression of hybrid PKS-NRPS and transporters promoted production of HYPs.

Discovery and biosynthesis of karnamicins as angiotensin converting enzyme inhibitors

Angiotensin-converting enzyme inhibitors are widely used for treatment of hypertension and related diseases. Here, six karnamicins E1-E6 (1–6), which bear fully substituted hydroxypyridine and thiazole moieties are characterized from the rare actinobacterium Lechevalieria rhizosphaerae NEAU-A2. Through a combination of isotopic labeling, genome mining, and enzymatic characterization studies, the programmed assembly of the fully substituted hydroxypyridine moiety in karnamicin is proposed to be due to sequential operation of a hybrid polyketide synthase-nonribosomal peptide synthetase, two regioselective pyridine ring flavoprotein hydroxylases, and a methyltransferase. Based on AlphaFold protein structures predictions, molecular docking, and site-directed mutagenesis, we find that two pyridine hydroxylases deploy active site residues distinct from other flavoprotein monooxygenases to direct the chemo- and regioselective hydroxylation of the pyridine nucleus. Pleasingly, karnamicins show significant angiotensin-converting enzyme inhibitory activity with IC50 values ranging from 0.24 to 5.81 μM, suggesting their potential use for the treatment of hypertension and related diseases.

Biosynthesis of Odd-Carbon Unsaturated Fatty Dicarboxylic Acids Through Engineering the HSAF Biosynthetic Gene in Lysobacter enzymogenes.

Fatty dicarboxylic acids (FDCA) are useful as starting materials or components for plastics, polyesters, nylons, and fragrances. Most of the commercially available FDCA contain an even number of carbons, and there remain few sustainable methods for production of FDCA with an odd number of carbons (o-FDCA). In this work, we explored a novel biosynthetic route to unsaturated o-FDCA. The approach was based on genetic modifications of hsaf pks-nrps, encoding a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) in Lysobacter enzymogenes, an environmental bacterium emerging as a new biocontrol agent. This single-module PKS-NRPS catalyzes the biosynthesis of lysobacterene A, a polyene-containing precursor of the antifungal natural product Heat-Stable Antifungal Factor (HSAF). We genetically removed the NRPS module from this gene and generated a new strain of L. enzymogenes, in which the PKS module was fused to the thioesterase domain of hsaf pks-nrps. The chimeric gene was verified by DNA sequencing, and its expression in L. enzymogenes was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The total fatty acids were extracted, esterified, and analyzed by GC-MS. The results showed that the engineered strain produced new fatty acids that were absent in the wild type. The main product was identified as hepta-2,4-dienedioic acid, an unsaturated o-FDCA. This work sets the foundation to explore a sustainable and environment-friendly approach toward unsaturated o-FDCA, which could be used as precursors for new compounds that can serve as versatile feedstock for industrial materials.

Tricarbocyclic core formation of tyrosine-decahydrofluorenes implies a three-enzyme cascade with XenF-mediated sigmatropic rearrangement as a prerequisite

Tyrosine-decahydrofluorene derivatives feature a fused [6.5.6] tricarbocyclic core and a 13-membered para-cyclophane ether. Herein, we identified new xenoacremones A, B, and C (1−3) from the fungal strain Xenoacremonium sinensis ML-31 and elucidated their biosynthetic pathway using gene deletion in the native strain and heterologous expression in Aspergillus nidulans. The hybrid polyketide synthase–nonribosomal peptide synthetase (PKS−NRPS) XenE together with enoyl reductase XenG were confirmed to be responsible for the formation of the tyrosine-nonaketide skeleton. This skeleton was subsequently dehydrated by XenA to afford a pyrrolidinone moiety. XenF catalyzed a novel sigmatropic rearrangement to yield a key cyclohexane intermediate as a prerequisite for the formation of the multi-ring system. Subsequent oxidation catalyzed by XenD supplied the substrate for XenC to link the para-cyclophane ether, which underwent subsequent spontaneous Diels−Alder reaction to give the end products. Thus, the results indicated that three novel enzymes XenF, XenD, and XenC coordinate to assemble the [6.5.6] tricarbocyclic ring and para-cyclophane ether during biosynthesis of complex tyrosine-decahydrofluorene derivatives.

Redox Modifications in the Biosynthesis of Alchivemycin A Enable the Formation of Its Key Pharmacophore.

Redox enzymes play a critical role in transforming nascent scaffolds into structurally complex and biologically active natural products. Alchivemycin A (AVM, 1) is a highly oxidized polycyclic compound with potent antimicrobial activity and features a rare 2H-tetrahydro-4,6-dioxo-1,2-oxazine (TDO) ring system. The scaffold of AVM has previously been shown to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway. In this study, we present a postassembly secondary metabolic network involving six redox enzymes that leads to AVM formation. We characterize this complex redox network using in vivo gene deletions, in vitro biochemical assays, and one-pot enzymatic total synthesis. Importantly, we show that an FAD-dependent monooxygenase catalyzes oxygen insertion into an amide bond to form the key TDO ring in AVM, an unprecedented function of flavoenzymes. We also show that the TDO ring is essential to the antimicrobial activity of AVM, likely through targeting the β-subunit of RNA polymerase. As further evidence, we show that AvmK, a β-subunit of RNA synthase, can confer self-resistance to AVM via target modification. Our findings expand the repertoire of functions of flavoenzymes and provide insight into antimicrobial and biocatalyst development based on AVM.

New Insight into Antimicrobial Compounds from Food and Marine-Sourced Carnobacterium Species through Phenotype and Genome Analyses.

Carnobacteriummaltaromaticum and Carnobacterium divergens, isolated from food products, are lactic acid bacteria known to produce active and efficient bacteriocins. Other species, particularly those originating from marine sources, are less studied. The aim of the study is to select promising strains with antimicrobial potential by combining genomic and phenotypic approaches on large datasets comprising 12 Carnobacterium species. The biosynthetic gene cluster (BGCs) diversity of 39 publicly available Carnobacterium spp. genomes revealed 67 BGCs, distributed according to the species and ecological niches. From zero to six BGCs were predicted per strain and classified into four classes: terpene, NRPS (non-ribosomal peptide synthetase), NRPS-PKS (hybrid non-ribosomal peptide synthetase-polyketide synthase), RiPP (ribosomally synthesized and post-translationally modified peptide). In parallel, the antimicrobial activity of 260 strains from seafood products was evaluated. Among the 60% of active strains, three genomes were sequenced and submitted to a dereplication process. C. inhibens MIP2551 produced a high amountof H2O2, probably thanks to the presence of four oxidase-encoding genes. C. maltaromaticum EBP3019 and SF668 strains were highly efficient against Listeria monocytogenes. A new extracellular 16 kDa unmodified bacteriocin in the EBP3019 strain and five different bacteriocins in SF668 were highlighted. In this study, the overview of antimicrobial BGC and inhibitory activities of Carnobacterium spp. allowed the prediction of potential innovative natural products that could be relevant for biotechnological applications.

Nonlinear Biosynthetic Assembly of Alpiniamide by a Hybrid cis/trans-AT PKS-NRPS.

Alpiniamide A is a linear polyketide produced by Streptomyces endophytic bacteria. Despite its relatively simple chemical structure suggestive of a linear assembly line biosynthetic construction involving a hybrid polyketide synthase-nonribosomal peptide synthetase enzymatic protein machine, we report an unexpected nonlinear synthesis of this bacterial natural product. Using a combination of genomics, heterologous expression, mutagenesis, isotope-labeling, and chain terminator experiments, we propose that alpiniamide A is assembled in two halves and then ligated into the mature molecule. We show that each polyketide half is constructed using orthogonal biosynthetic strategies, employing either cis- or trans-acyl transferase mechanisms, thus prompting an alternative proposal for the operation of this PKS-NRPS.

Characterization of Miharamycin Biosynthesis Reveals a Hybrid NRPS-PKS to Synthesize High-Carbon Sugar from a Complex Nucleoside.

Miharamycins are peptidyl nucleoside antibiotics with a unique branched C9 pyranosyl amino acid core and a rare 2-aminopurine moiety. Inactivation of 19 genes in the biosynthetic gene cluster and identification of several unexpected intermediates suggest an alternative biosynthetic pathway, which is further supported by feeding experiments and in vitro characterization of an unusual adenylation domain recognizing a complex nucleoside derivative as the substrate. These results thereby provide an unprecedented biosynthetic route of high-carbon sugar catalyzed by atypical hybrid nonribosomal peptide synthetase-polyketide synthase.

Genus level analysis of PKS-NRPS and NRPS-PKS hybrids reveals their origin in Aspergilli

BackgroundFilamentous fungi produce a vast amount of bioactive secondary metabolites (SMs) synthesized by e.g. hybrid polyketide synthase-nonribosomal peptide synthetase enzymes (PKS-NRPS; NRPS-PKS). While their domain structure suggests a common ancestor with other SM proteins, their evolutionary origin and dynamics in fungi are still unclear. Recent rational engineering approaches highlighted the possibility to reassemble hybrids into chimeras — suggesting molecular recombination as diversifying mechanism.ResultsPhylogenetic analysis of hybrids in 37 species – spanning 9 sections of Aspergillus and Penicillium chrysogenum – let us describe their dynamics throughout the genus Aspergillus. The tree topology indicates that three groups of PKS-NRPS as well as one group of NRPS-PKS hybrids developed independently from each other. Comparison to other SM genes lead to the conclusion that hybrids in Aspergilli have several PKS ancestors; in contrast, hybrids are monophyletic when compared to available NRPS genes — with the exception of a small group of NRPSs. Our analysis also revealed that certain NRPS-likes are derived from NRPSs, suggesting that the NRPS/NRPS-like relationship is dynamic and proteins can diverge from one function to another. An extended phylogenetic analysis including bacterial and fungal taxa revealed multiple ancestors of hybrids. Homologous hybrids are present in all sections which suggests frequent horizontal gene transfer between genera and a finite number of hybrids in fungi.ConclusionPhylogenetic distances between hybrids provide us with evidence for their evolution: Large inter-group distances indicate multiple independent events leading to the generation of hybrids, while short intra-group distances of hybrids from different taxonomic sections indicate frequent horizontal gene transfer. Our results are further supported by adding bacterial and fungal genera. Presence of related hybrid genes in all Ascomycetes suggests a frequent horizontal gene transfer between genera and a finite diversity of hybrids — also explaining their scarcity. The provided insights into relations of hybrids and other SM genes will serve in rational design of new hybrid enzymes.

Discovery and Characterization of 1-Aminocyclopropane-1-carboxylic Acid Synthase of Bacterial Origin.

The guangnanmycins (GNMs) belong to a small group of natural products featuring a 1-aminocyclopropane-1-carboxylic acid (ACC) moiety. While extensively studied in plants, ACC biosynthesis in bacteria remains poorly understood. Here we report inactivation of gnmY in vivo and biochemical characterization of GnmY in vitro, assigning GnmY as the first bacterial free ACC synthase that catalyzes the synthesis of ACC from S-adenosyl methionine. ACC is activated by GnmS and subsequently incorporated into the GNM scaffold by the GNM hybrid nonribosomal peptide synthetase-polyketide synthase system in GNM biosynthesis. GnmS exhibits relaxed substrate specificity, exploitation of which allowed the incorporation of 1-aminocyclobutane-1-carboxylic acid (ACBC) into the GNM scaffold to produce a GNM analogue with a cyclobutane ring at C-17. This study provides new insights into ACC biosynthesis in bacteria. GnmY and GnmS might be portable to engineer other ACC/ACBC-containing natural products.

Ralstonins A and B, Lipopeptides with Chlamydospore-Inducing and Phytotoxic Activities from the Plant Pathogen Ralstonia solanacearum.

Ralstonia solanacearum has an orphan hybrid polyketide synthase-nonribosomal peptide synthetase gene cluster. We herein isolate its products (named ralstonins A and B) from R. solanacearum and elucidate their structures and biological activities. Ralstonins are unusual lipodepsipeptides composed of 11 amino acids (containing unique amino acids such as β-hydroxytyrosine and dehydroalanine) and a 3-amino-2-hydroxyoctadecanoic acid, and their production is controlled by quorum sensing, a mechanism of bacterial cell-cell communication. Ralstonins exhibited chlamydospore-inducing activity and phytotoxicity.

A scalable platform to identify fungal secondary metabolites and their gene clusters.

The genomes of filamentous fungi contain up to ~90 biosynthetic gene clusters (BGCs), encoding diverse secondary metabolites, an enormous reservoir of untapped chemical potential. However, recalcitrant genetics, cryptic expression, and unculturability prevent the systematic exploitation of these gene clusters and harvesting of their products. With heterologous expression of fungal BGCs largely limited to expression of single or partial clusters, we established a scalable process for expression of large numbers of full-length gene clusters, called FAC-MS. Using Fungal Artificial Chromosomes (FACs) with Metabolomic Scoring (MS) we screened 56 secondary metabolite BGCs from diverse fungal species for expression in A. nidulans. Fifteen new metabolites were discovered and confidently assigned to their BGCs. A new macrolactone, valactamide A, and its hybrid PKS-NRPS gene cluster were characterized extensively using this integrated platform. Regularizing access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues.

Ralstonia solanacearum is a globally distributed soil-borne plant pathogenic bacterium, which shares a broad ecological range with many plant- and soil-associated fungi. We sought to determine if R. solanacearum chemical communication directs symbiotic development of polymicrobial consortia. R. solanacearum produced a diffusible metabolite that induced conserved morphological differentiation in 34 species of fungi across three diverse taxa (Ascomycetes, Basidiomycetes and Zygomycetes). Fungi exposed to this metabolite formed chlamydospores, survival structures with thickened cell walls. Some chlamydospores internally harbored R. solanacearum, indicating a newly described endofungal lifestyle for this important plant pathogen. Using imaging mass spectrometry and peptidogenomics, we identified an undescribed lipopeptide, ralsolamycin, produced by an R. solanacearum non-ribosomal peptide synthetase-polyketide synthase hybrid. Inactivation of the hybrid non-ribosomal peptide synthetase-polyketide synthase gene, rmyA, abolished ralsolamycin synthesis. R. solanacearum mutants lacking ralsolamycin no longer induced chlamydospore development in fungal coculture and invaded fungal hyphae less well than wild-type. We propose that ralsolamycin contributes to the invasion of fungal hyphae and that the formation of chlamydospores may provide not only a specific niche for bacterial colonization but also enhanced survival for the partnering fungus.

Characterization of Polyketide Synthase Machinery from the pks Island Facilitates Isolation of a Candidate Precolibactin.

Colibactin is a human gut bacterial genotoxin of unknown structure that has been linked to colon cancer. The biosynthesis of this elusive metabolite is directed by the pks gene cluster, which encodes a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line that is hypothesized to use the unusual polyketide building block aminomalonate. This biosynthetic pathway is thought to initially produce an inactive intermediate (precolibactin) that is processed to the active toxin. Here, we report the first in vitro biochemical characterization of the PKS components of the pks enzymatic assembly line. We evaluate PKS extender unit utilization and show that ClbG, a freestanding acyltransferase (AT) from the pks gene cluster, recognizes aminomalonyl-acyl carrier protein (AM-ACP) and transfers this building block to multiple PKS modules, including a cis-AT PKS ClbI. We also use genetics to explore the in vivo role of ClbG in colibactin and precolibactin biosynthesis. Unexpectedly, production of previously identified pks-associated metabolites is dramatically increased in a ΔclbP/ΔclbG mutant strain, enabling the first structure elucidation of a bithiazole-containing candidate precolibactin. This work provides new insights into the unusual biosynthetic capabilities of the pks gene cluster, offers further support for the hypothesis that colibactin directly damages DNA, and suggests that additional, uncharacterized pks-derived metabolites containing aminomalonate play critical roles in genotoxicity.

Multifunctional polyketide synthase genes identified by genomic survey of the symbiotic dinoflagellate, Symbiodinium minutum.

BackgroundDinoflagellates are unicellular marine and freshwater eukaryotes. They possess large nuclear genomes (1.5–245 gigabases) and produce structurally unique and biologically active polyketide secondary metabolites. Although polyketide biosynthesis is well studied in terrestrial and freshwater organisms, only recently have dinoflagellate polyketides been investigated. Transcriptomic analyses have characterized dinoflagellate polyketide synthase genes having single domains. The Genus Symbiodinium, with a comparatively small genome, is a group of major coral symbionts, and the S. minutum nuclear genome has been decoded.ResultsThe present survey investigated the assembled S. minutum genome and identified 25 candidate polyketide synthase (PKS) genes that encode proteins with mono- and multifunctional domains. Predicted proteins retain functionally important amino acids in the catalytic ketosynthase (KS) domain. Molecular phylogenetic analyses of KS domains form a clade in which S. minutum domains cluster within the protist Type I PKS clade with those of other dinoflagellates and other eukaryotes. Single-domain PKS genes are likely expanded in dinoflagellate lineage. Two PKS genes of bacterial origin are found in the S. minutum genome. Interestingly, the largest enzyme is likely expressed as a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly of 10,601 amino acids, containing NRPS and PKS modules and a thioesterase (TE) domain. We also found intron-rich genes with the minimal set of catalytic domains needed to produce polyketides. Ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) along with other optional domains are present. Mapping of transcripts to the genome with the dinoflagellate-specific spliced leader sequence, supports expression of multifunctional PKS genes. Metabolite profiling of cultured S. minutum confirmed production of zooxanthellamide D, a polyhydroxy amide polyketide and other unknown polyketide secondary metabolites.ConclusionThis genomic survey demonstrates that S. minutum contains genes with the minimal set of catalytic domains needed to produce polyketides and provides evidence of the modular nature of Type I PKS, unlike monofunctional Type I PKS from other dinoflagellates. In addition, our study suggests that diversification of dinoflagellate PKS genes comprises dinoflagellate-specific PKS genes with single domains, multifunctional PKS genes with KS domains orthologous to those of other protists, and PKS genes of bacterial origin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2195-8) contains supplementary material, which is available to authorized users.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS–NRPS is necessary for synthesis of the 2-pyridones, leporins

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS–non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange–red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea

Fumosorinone, isolated from the entomogenous fungus Isaria fumosorosea, is a new 2-pyridone alkaloid which is elucidated by HRESIMS 1D and 2DNMR. Fumosorinone is structurally similar to tenellin and desmethylbassianin but it differs in chain length and degree of methylation. It is characterized by a classic noncompetitive inhibitor of protein tyrosine phosphatase 1B (IC50 14.04μM) which was implicated as a negative regulator of insulin receptor signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. For further study, we identified the biosynthetic gene cluster of fumosorinone from ongoing genome sequencing project, and it was verified by a direct knock-out strategy, reported for the first time in I. fumosorosea, using the Agrobacterium-mediated transformation in conjunction with linear deletion cassettes. The biosynthetic gene cluster includes a hybrid polyketide synthase–nonribosomal peptide synthetase gene, two cytochrome P450 enzyme genes, a trans-enoyl reductase gene, and other two transcription regulatory genes. Comparison of fumosorinone biosynthetic cluster with known gene clusters gives further insight into biosynthesis of pyridone alkaloids and provides the foundation for combinatorial biosynthesis for new fumosorinone derivatives.

Spliceostatin hemiketal biosynthesis in Burkholderia spp. is catalyzed by an iron/α-ketoglutarate-dependent dioxygenase.

Spliceostatins are potent spliceosome inhibitors biosynthesized by a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) system of the trans-acyl transferase (AT) type. Burkholderia sp. FERM BP-3421 produces hemiketal spliceostatins, such as FR901464, as well as analogs containing a terminal carboxylic acid. We provide genetic and biochemical evidence for hemiketal biosynthesis by oxidative decarboxylation rather than the previously hypothesized Baeyer-Villiger oxidative release postulated to be catalyzed by a flavin-dependent monooxygenase (FMO) activity internal to the last module of the PKS. Inactivation of Fe(II)/α-ketoglutarate-dependent dioxygenase gene fr9P led to loss of hemiketal congeners, whereas the mutant was still able to produce all major carboxylic acid-type compounds. FMO mutants, on the other hand, produced both hemiketal and carboxylic acid analogs containing an exocyclic methylene instead of an epoxide, indicating that the FMO is involved in epoxidation rather than Baeyer-Villiger oxidation. Moreover, recombinant Fr9P enzyme was shown to catalyze hydroxylation to form β-hydroxy acids, which upon decarboxylation led to hemiketal FR901464. Finally, a third oxygenase activity encoded in the biosynthetic gene cluster, the cytochrome P450 monooxygenase Fr9R, was assigned as a 4-hydroxylase based on gene inactivation results. Identification and deletion of the gene involved in hemiketal formation allowed us to generate a strain--the dioxygenase fr9P(-) mutant--that accumulates only the carboxylic acid-type spliceostatins, which are as potent as the hemiketal analogs, when derivatized to increase cell permeability, but are chemically more stable.