DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirimura, Kohtaro, E-mail: kkohtaro@waseda.jp; Watanabe, Shotaro; Kobayashi, Keiichi

Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr-8-2: 2978617–2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni{sup 2+}-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity ofmore » the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. -- Highlights: •Type III PKS from Aspergillus niger NRRL 328, An-CsyA, was cloned and characterized. •An-CsyA produced triketide pyrones, tetraketide pyrones and pentaketide resorcinols. •Functional properties of An-CsyA differs from those of other fungal type III PKSs.« less

Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I modular polyketide synthases: opportunities and challenges.

PubMed

Yuzawa, Satoshi; Keasling, Jay D; Katz, Leonard

2017-04-01

Complex polyketides comprise a large number of natural products that have broad application in medicine and agriculture. They are produced in bacteria and fungi from large enzyme complexes named type I modular polyketide synthases (PKSs) that are composed of multifunctional polypeptides containing discrete enzymatic domains organized into modules. The modular nature of PKSs has enabled a multitude of efforts to engineer the PKS genes to produce novel polyketides of predicted structure. We have repurposed PKSs to produce a number of short-chain mono- and di-carboxylic acids and ketones that could have applications as fuels or industrial chemicals.

The industrial anaerobe Clostridium acetobutylicum uses polyketides to regulate cellular differentiation.

PubMed

Herman, Nicolaus A; Kim, Seong Jong; Li, Jeffrey S; Cai, Wenlong; Koshino, Hiroyuki; Zhang, Wenjun

2017-11-15

Polyketides are an important class of bioactive small molecules valued not only for their diverse therapeutic applications, but also for their role in controlling interesting biological phenotypes in their producing organisms. While numerous polyketides are known to be derived from aerobic organisms, only a single family of polyketides has been identified from anaerobic organisms. Here we uncover a family of polyketides native to the anaerobic bacterium Clostridium acetobutylicum, an organism well-known for its historical use as an industrial producer of the organic solvents acetone, butanol, and ethanol. Through mutational analysis and chemical complementation assays, we demonstrate that these polyketides act as chemical triggers of sporulation and granulose accumulation in this strain. This study represents a significant addition to the body of work demonstrating the existence and importance of polyketides in anaerobes, and showcases a strategy of manipulating the secondary metabolism of an organism to improve traits relevant for industrial applications.

Cloning, sequencing, and analysis of the griseusin polyketide synthase gene cluster from Streptomyces griseus.

PubMed Central

Yu, T W; Bibb, M J; Revill, W P; Hopwood, D A

1994-01-01

A fragment of DNA was cloned from the Streptomyces griseus K-63 genome by using genes (act) for the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor as a probe. Sequencing of a 5.4-kb segment of the cloned DNA revealed a set of five gris open reading frames (ORFs), corresponding to the act PKS genes, in the following order: ORF1 for a ketosynthase, ORF2 for a chain length-determining factor, ORF3 for an acyl carrier protein, ORF5 for a ketoreductase, and ORF4 for a cyclase-dehydrase. Replacement of the gris genes with a marker gene in the S. griseus genome by using a single-stranded suicide vector propagated in Escherichia coli resulted in loss of the ability to produce griseusins A and B, showing that the five gris genes do indeed encode the type II griseusin PKS. These genes, encoding a PKS that is programmed differently from those for other aromatic PKSs so far available, will provide further valuable material for analysis of the programming mechanism by the construction and analysis of strains carrying hybrid PKS. Images PMID:8169211

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuzawa, Satoshi; Keasling, Jay D.; Katz, Leonard

Complex polyketides comprise a large number of natural products that have broad application in medicine and agriculture. They are produced in bacteria and fungi from large enzyme complexes named type I modular polyketide synthases (PKSs) that are composed of multifunctional polypeptides containing discrete enzymatic domains organized into modules. The modular nature of PKSs has enabled a multitude of efforts to engineer the PKS genes to produce novel polyketides of predicted structure. Finally, we have repurposed PKSs to produce a number of short-chain mono- and di-carboxylic acids and ketones that could have applications as fuels or industrial chemicals.

Algal carbohydrates affect polyketide synthesis of the lichen-forming fungus Cladonia rangiferina.

PubMed

Elshobary, Mostafa E; Osman, Mohamed E; Abo-Shady, Atef M; Komatsu, Emy; Perreault, Hélène; Sorensen, John; Piercey-Normore, Michele D

2016-01-01

Lichen secondary metabolites (polyketides) are produced by the fungal partner, but the role of algal carbohydrates in polyketide biosynthesis is not clear. This study examined whether the type and concentration of algal carbohydrate explained differences in polyketide production and gene transcription by a lichen fungus (Cladonia rangiferina). The carbohydrates identified from a free-living cyanobacterium (Spirulina platensis; glucose), a lichen-forming alga (Diplosphaera chodatii; sorbitol) and the lichen alga that associates with C. rangiferina (Asterochloris sp.; ribitol) were used in each of 1%, 5% and 10% concentrations to enrich malt yeast extract media for culturing the mycobiont. Polyketides were determined by high performance liquid chromatography (HPLC), and polyketide synthase (PKS) gene transcription was measured by quantitative PCR of the ketosynthase domain of four PKS genes. The lower concentrations of carbohydrates induced the PKS gene expression where ribitol up-regulated CrPKS1 and CrPKS16 gene transcription and sorbitol up-regulated CrPKS3 and CrPKS7 gene transcription. The HPLC results revealed that lower concentrations of carbon sources increased polyketide production for three carbohydrates. One polyketide from the natural lichen thallus (fumarprotocetraric acid) also was produced by the fungal culture in ribitol supplemented media only. This study provides a better understanding of the role of the type and concentration of the carbon source in fungal polyketide biosynthesis in the lichen Cladonia rangiferina. © 2016 by The Mycological Society of America.

Metabolomic and Transcriptomic Comparison of Solid-State and Submerged Fermentation of Penicillium expansum KACC 40815

PubMed Central

Park, Hye Min; Singh, Digar; Lee, Choong Hwan

2016-01-01

Penicillium spp. are known to harbor a wide array of secondary metabolites with cryptic bioactivities. However, the metabolomics of these species is not well-understood in terms of different fermentation models and conditions. The present study involved metabolomics profiling and transcriptomic analysis of Penicillium expansum 40815 under solid-state fermentation (SSF) and submerged fermentation (SmF). Metabolite profiling was carried out using ultra-performance liquid chromatography quadruple time-of-flight mass spectrometry with multivariate analysis, followed by transcriptomic analyses of differentially expressed genes. In principal component analysis, the metabolite profiling data was studied under different experimental sets, including SSF and SmF. The significantly different metabolites such as polyketide metabolites (agonodepside B, rotiorin, verrucosidin, and ochrephilone) and corresponding gene transcripts (polyketide synthase, aromatic prenyltransferase, and terpenoid synthase) were primarily detected under SmF conditions. In contrast, the meroterpenoid compounds (andrastin A and C) and their genes transcripts were exclusively detected under SSF conditions. We demonstrated that the metabolite production and its corresponding gene expression levels in P. expansum 40815 were significantly influenced by the varying growth parameters and the immediate environment. This study further provides a foundation to produce specific metabolites by regulating fermentation conditions. PMID:26863302

Metabolomic and Transcriptomic Comparison of Solid-State and Submerged Fermentation of Penicillium expansum KACC 40815.

PubMed

Kim, Hyang Yeon; Heo, Do Yeon; Park, Hye Min; Singh, Digar; Lee, Choong Hwan

2016-01-01

Penicillium spp. are known to harbor a wide array of secondary metabolites with cryptic bioactivities. However, the metabolomics of these species is not well-understood in terms of different fermentation models and conditions. The present study involved metabolomics profiling and transcriptomic analysis of Penicillium expansum 40815 under solid-state fermentation (SSF) and submerged fermentation (SmF). Metabolite profiling was carried out using ultra-performance liquid chromatography quadruple time-of-flight mass spectrometry with multivariate analysis, followed by transcriptomic analyses of differentially expressed genes. In principal component analysis, the metabolite profiling data was studied under different experimental sets, including SSF and SmF. The significantly different metabolites such as polyketide metabolites (agonodepside B, rotiorin, verrucosidin, and ochrephilone) and corresponding gene transcripts (polyketide synthase, aromatic prenyltransferase, and terpenoid synthase) were primarily detected under SmF conditions. In contrast, the meroterpenoid compounds (andrastin A and C) and their genes transcripts were exclusively detected under SSF conditions. We demonstrated that the metabolite production and its corresponding gene expression levels in P. expansum 40815 were significantly influenced by the varying growth parameters and the immediate environment. This study further provides a foundation to produce specific metabolites by regulating fermentation conditions.

Polyketide synthesis genes associated with toxin production in two species of Gambierdiscus (Dinophyceae).

PubMed

Kohli, Gurjeet S; John, Uwe; Figueroa, Rosa I; Rhodes, Lesley L; Harwood, D Tim; Groth, Marco; Bolch, Christopher J S; Murray, Shauna A

2015-05-28

Marine microbial protists, in particular, dinoflagellates, produce polyketide toxins with ecosystem-wide and human health impacts. Species of Gambierdiscus produce the polyether ladder compounds ciguatoxins and maitotoxins, which can lead to ciguatera fish poisoning, a serious human illness associated with reef fish consumption. Genes associated with the biosynthesis of polyether ladder compounds are yet to be elucidated, however, stable isotope feeding studies of such compounds consistently support their polyketide origin indicating that polyketide synthases are involved in their biosynthesis. Here, we report the toxicity, genome size, gene content and transcriptome of Gambierdiscus australes and G. belizeanus. G. australes produced maitotoxin-1 and maitotoxin-3, while G. belizeanus produced maitotoxin-3, for which cell extracts were toxic to mice by IP injection (LD50 = 3.8 mg kg(-1)). The gene catalogues comprised 83,353 and 84,870 unique contigs, with genome sizes of 32.5 ± 3.7 Gbp and 35 ± 0.88 Gbp, respectively, and are amongst the most comprehensive yet reported from a dinoflagellate. We found three hundred and six genes involved in polyketide biosynthesis, including one hundred and ninety-two ketoacyl synthase transcripts, which formed five unique phylogenetic clusters. Two clusters were unique to these maitotoxin-producing dinoflagellate species, suggesting that they may be associated with maitotoxin biosynthesis. This work represents a significant step forward in our understanding of the genetic basis of polyketide production in dinoflagellates, in particular, species responsible for ciguatera fish poisoning.

Producing alpha-olefins using polyketide synthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fortman, Jeffrey L.; Katz, Leonard; Steen, Eric J.

2018-01-02

The present invention provides for a polyketide synthase (PKS) capable of synthesizing an .alpha.-olefin, such as 1-hexene or butadiene. The present invention also provides for a host cell comprising the PKS and when cultured produces the .alpha.-olefin.

Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

PubMed Central

Kumano, Takuto; Richard, Stéphane B.; Noel, Joseph P.; Nishiyama, Makoto; Kuzuyama, Tomohisa

2010-01-01

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon–carbon-based and carbon–oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism. PMID:18682327

The Distant Siblings-A Phylogenomic Roadmap Illuminates the Origins of Extant Diversity in Fungal Aromatic Polyketide Biosynthesis.

PubMed

Koczyk, Grzegorz; Dawidziuk, Adam; Popiel, Delfina

2015-11-03

In recent years, the influx of newly sequenced fungal genomes has enabled sampling of secondary metabolite biosynthesis on an unprecedented scale. However, explanations of extant diversity which take into account both large-scale phylogeny reconstructions and knowledge gained from multiple genome projects are still lacking. We analyzed the evolutionary sources of genetic diversity in aromatic polyketide biosynthesis in over 100 model fungal genomes. By reconciling the history of over 400 nonreducing polyketide synthases (NR-PKSs) with corresponding species history, we demonstrate that extant fungal NR-PKSs are clades of distant siblings, originating from a burst of duplications in early Pezizomycotina and thinned by extensive losses. The capability of higher fungi to biosynthesize the simplest precursor molecule (orsellinic acid) is highlighted as an ancestral trait underlying biosynthesis of aromatic compounds. This base activity was modified during early evolution of filamentous fungi, toward divergent reaction schemes associated with biosynthesis of, for example, aflatoxins and fusarubins (C4-C9 cyclization) or various anthraquinone derivatives (C6-C11 cyclization). The functional plasticity is further shown to have been supplemented by modularization of domain architecture into discrete pieces (conserved splice junctions within product template domain), as well as tight linkage of key accessory enzyme families and divergence in employed transcriptional factors. Although the majority of discord between species and gene history is explained by ancient duplications, this landscape has been altered by more recent duplications, as well as multiple horizontal gene transfers. The 25 detected transfers include previously undescribed events leading to emergence of, for example, fusarubin biosynthesis in Fusarium genus. Both the underlying data and the results of present analysis (including alternative scenarios revealed by sampling multiple reconciliation optima) are maintained as a freely available web-based resource: http://cropnet.pl/metasites/sekmet/nrpks_2014. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Producing dicarboxylic acids using polyketide synthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katz, Leonard; Fortman, Jeffrey L.; Keasling, Jay D.

The present invention provides for a polyketide synthase (PKS) capable of synthesizing a dicarboxylic acid (diacid). Such diacids include diketide-diacids and triketide-diacids. The invention includes recombinant nucleic acid encoding the PKS, and host cells comprising the PKS. The invention also includes methods for producing the diacids.

Enabling techniques in the search for new antibiotics: Combinatorial biosynthesis of sugar-containing antibiotics.

PubMed

Park, Je Won; Nam, Sang-Jip; Yoon, Yeo Joon

2017-06-15

Nature has a talent for inventing a vast number of natural products, including hybrids generated by blending different scaffolds, resulting in a myriad of bioactive chemical entities. Herein, we review the highlights and recent trends (2010-2016) in the combinatorial biosynthesis of sugar-containing antibiotics where nature's structural diversification capabilities are exploited to enable the creation of new anti-infective and anti-proliferative drugs. In this review, we describe the modern combinatorial biosynthetic approaches for polyketide synthase-derived complex and aromatic polyketides, non-ribosomal peptide synthetase-directed lipo-/glycopeptides, aminoglycosides, nucleoside antibiotics, and alkaloids, along with their therapeutic potential. Finally, we present the feasible nexus between combinatorial biosynthesis, systems biology, and synthetic biology as a toolbox to provide new antibiotics that will be indispensable in the post-antibiotic era. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential Expression of Putative Polyketide Biosynthetic Gene Clusters in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

The maize pathogen Fusarium verticillioides can produce a number of polyketide derived secondary metabolites, including fumonisins. Fumonisins cause diseases in animals, and show epidemiological correlation with esophageal cancer and birth defects in humans. The F. verticillioides genome contains ...

Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from Streptomyces rimosus NRRL 3016.

PubMed Central

Hong, S T; Carney, J R; Gould, S J

1997-01-01

The genes for the complete pathways for two polycyclic aromatic polyketides of the angucyclinone class have been cloned and heterologously expressed. Genomic DNAs of Streptomyces rimosus NRRL 3016 and Streptomyces strain WP 4669 were partially digested with MboI, and libraries (ca. 40-kb fragments) in Escherichia coli XL1-Blue MR were prepared with the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes from each organism. After transfer of the four clusters to Streptomyces lividans TK24, expression of one cluster from each organism was established through the identification of pathway-specific products by high-performance liquid chromatography with photodiode array detection. Peaks were identified from the S. rimosus cluster (pksRIM-1) for tetrangulol, tetrangomycin, and fridamycin E. Peaks were identified from the WP 4669 cluster (pksWP-2) for tetrangulol, 19-hydroxytetrangulol, 8-O-methyltetrangulol, 19-hydroxy-8-O-methyltetrangulol, and PD 116740. Structures were confirmed by 1H nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. PMID:8990300

Cloning and heterologous expression of the entire gene clusters for PD 116740 from Streptomyces strain WP 4669 and tetrangulol and tetrangomycin from Streptomyces rimosus NRRL 3016.

PubMed

Hong, S T; Carney, J R; Gould, S J

1997-01-01

The genes for the complete pathways for two polycyclic aromatic polyketides of the angucyclinone class have been cloned and heterologously expressed. Genomic DNAs of Streptomyces rimosus NRRL 3016 and Streptomyces strain WP 4669 were partially digested with MboI, and libraries (ca. 40-kb fragments) in Escherichia coli XL1-Blue MR were prepared with the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes from each organism. After transfer of the four clusters to Streptomyces lividans TK24, expression of one cluster from each organism was established through the identification of pathway-specific products by high-performance liquid chromatography with photodiode array detection. Peaks were identified from the S. rimosus cluster (pksRIM-1) for tetrangulol, tetrangomycin, and fridamycin E. Peaks were identified from the WP 4669 cluster (pksWP-2) for tetrangulol, 19-hydroxytetrangulol, 8-O-methyltetrangulol, 19-hydroxy-8-O-methyltetrangulol, and PD 116740. Structures were confirmed by 1H nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry.

Exploration of industrially important pigments from soil fungi.

PubMed

Akilandeswari, P; Pradeep, B V

2016-02-01

The worldwide interest of the current era is to increase tendency towards the use of natural substances instead of synthetic ones. So, alternative and effective environment friendly sustainable technologies are highly needed. Due to a broad range of biological activities, fungi are considered as a significant source of pigments. Among the fungal species in the soil, the genera of Aspergillus, Fusarium, Penicillium, Paecilomyces, and Trichoderma are dominant. The pigments commonly produced by fungi belong to aromatic polyketide groups such as melanins, quinones, flavins, ankaflavin, anthraquinone, and naphthoquinone. The use of fungal pigments has benefits which comprise easy and fast growth in the cheap culture medium and different color shades being independent of weather conditions and would be useful in various industrial applications. In relation to the toxic effects of the synthetic dyes, the natural dyes are easily degradable since they cause no detrimental effects. Thus, the study of pigments produced by soil fungi has tremendous use in medical, textile coloring, food coloring, and cosmetics.

Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL.

PubMed

Kalaitzis, John A; Cheng, Qian; Meluzzi, Dario; Xiang, Longkuan; Izumikawa, Miho; Dorrestein, Pieter C; Moore, Bradley S

2011-11-15

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete 'Streptomyces maritimus'. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-β-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates. Copyright © 2011 Elsevier Ltd. All rights reserved.

Policing Starter Unit Selection of the Enterocin Type II Polyketide Synthase by the Type II Thioesterase EncL

PubMed Central

Kalaitzis, John A.; Cheng, Qian; Meluzzi, Dario; Xiang, Longkuan; Izumikawa, Miho; Dorrestein, Pieter C.; Moore, Bradley S.

2011-01-01

Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete “Streptomyces maritimus”. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-β-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates. PMID:21531566

EncM, a versatile enterocin biosynthetic enzyme involved in Favorskii oxidative rearrangement, aldol condensation, and heterocycle-forming reactions

PubMed Central

Xiang, Longkuan; Kalaitzis, John A.; Moore, Bradley S.

2004-01-01

The bacteriostatic natural product enterocin from the marine microbe “Streptomyces maritimus” has an unprecedented carbon skeleton that is derived from an aromatic polyketide biosynthetic pathway. Its caged tricyclic, nonaromatic core is derived from a linear poly-β-ketide precursor that formally undergoes a Favorskii-like oxidative rearrangement. In vivo characterization of the gene encM through mutagenesis and heterologous biosynthesis demonstrated that its protein product not only is solely responsible for the oxidative C—C rearrangement, but also facilitates two aldol condensations plus two heterocycle forming reactions. In total, at least five chiral centers and four rings are generated by this multifaceted flavoprotein. Heterologous expression of the enterocin biosynthesis genes encABCDLMN in Streptomyces lividans resulted in the formation of the rearranged metabolite desmethyl-5-deoxyenterocin and the shunt products wailupemycins D-G. Addition of the methyltransferase gene encK, which was previously proposed through mutagenesis to additionally assist EncM in the Favorskii rearrangement, shifted the production to the O-methyl derivative 5-deoxyenterocin. The O-methyltransferase EncK seems to be specific for the pyrone ring of enterocin, because bicyclic polyketides bearing pyrone rings are not methylated in vivo. Expression of encM with different combinations of homologous actinorhodin biosynthesis genes did not result in the production of oxidatively rearranged enterocin-actinorhodin hybrid compounds as anticipated, suggesting that wild-type EncM may be specific for its endogenous type II polyketide synthase or for benzoyl-primed polyketide precursors. PMID:15505225

Producing a trimethylpentanoic acid using hybrid polyketide synthases

DOEpatents

Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

2014-10-07

The present invention provides for a polyketide synthase (PKS) capable of synthesizing trimethylpentanoic acid. The present invention also provides for a host cell comprising the PKS and when cultured produces the trimethylpentanoic acid. The present invention also provides for a method of producing the trimethylpentanoic acid, comprising: providing a host cell of the present invention, and culturing said host cell in a suitable culture medium such that the trimethylpentanoic acid is produced, optionally isolating the trimethylpentanoic acid, and optionally, reducing the isolated trimethylpentanoic acid into a trimethylpentanol or an iso-octane.

Hybrid polyketide synthases

DOEpatents

Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

2016-05-10

The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria

USDA-ARS?s Scientific Manuscript database

Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small mole...

Saccharomyces cerevisiae as a tool for mining, studying and engineering fungal polyketide synthases

PubMed Central

Bond, Carly; Tang, Yi; Li, Li

2016-01-01

Small molecule secondary metabolites produced by organisms such as plants, bacteria, and fungi form a fascinating and important group of natural products, many of which have shown promise as medicines. Fungi in particular have been important sources of natural product polyketide pharmaceuticals. While the structural complexity of these polyketides makes them interesting and useful bioactive compounds, these same features also make them difficult and expensive to prepare and scale-up using synthetic methods. Currently, nearly all commercial polyketides are prepared through fermentation or semi-synthesis. However, elucidation and engineering of polyketide pathways in the native filamentous fungi hosts are often hampered due to a lack of established genetic tools and of understanding of the regulation of fungal secondary metabolisms. Saccharomyces cerevisiae has many advantages beneficial to the study and development of polyketide pathways from filamentous fungi due to its extensive genetic toolbox and well-studied metabolism. This review highlights the benefits S. cerevisiae provides as a tool for mining, studying, and engineering fungal polyketide synthases (PKSs), as well as notable insights this versatile tool has given us into the mechanisms and products of fungal PKSs. PMID:26850128

Saccharomyces cerevisiae as a tool for mining, studying and engineering fungal polyketide synthases.

PubMed

Bond, Carly; Tang, Yi; Li, Li

2016-04-01

Small molecule secondary metabolites produced by organisms such as plants, bacteria, and fungi form a fascinating and important group of natural products, many of which have shown promise as medicines. Fungi in particular have been important sources of natural product polyketide pharmaceuticals. While the structural complexity of these polyketides makes them interesting and useful bioactive compounds, these same features also make them difficult and expensive to prepare and scale-up using synthetic methods. Currently, nearly all commercial polyketides are prepared through fermentation or semi-synthesis. However, elucidation and engineering of polyketide pathways in the native filamentous fungi hosts are often hampered due to a lack of established genetic tools and of understanding of the regulation of fungal secondary metabolisms. Saccharomyces cerevisiae has many advantages beneficial to the study and development of polyketide pathways from filamentous fungi due to its extensive genetic toolbox and well-studied metabolism. This review highlights the benefits S. cerevisiae provides as a tool for mining, studying, and engineering fungal polyketide synthases (PKSs), as well as notable insights this versatile tool has given us into the mechanisms and products of fungal PKSs. Copyright © 2016 Elsevier Inc. All rights reserved.

Fumonisin-nonproducing mutants exhibit differential expression of putative polyketide biosynthetic gene clusters in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

The maize pathogen Fusarium verticillioides produces a group of polyketide derived secondary metabolites called fumonisins. Fumonisins can cause diseases in animals, and have been correlated epidemiologically with esophageal cancer and birth defects in humans. The fumonisin biosynthetic gene clust...

Altered expression of polyketide biosynthetic gene clusters in fumonisin-deficient mutants of Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

Fusarium verticillioides is a pathogen of maize and produces fumonisins, a group of polyketide derived secondary metabolites. Fumonisins cause diseases in animals, and they have been correlated epidemiologically with esophageal cancer and birth defects in humans. Fumonisin biosynthetic genes are c...

A unifying paradigm for naphthoquinone-based meroterpenoid (bio)synthesis

NASA Astrophysics Data System (ADS)

Miles, Zachary D.; Diethelm, Stefan; Pepper, Henry P.; Huang, David M.; George, Jonathan H.; Moore, Bradley S.

2017-12-01

Bacterial meroterpenoids constitute an important class of natural products with diverse biological properties and therapeutic potential. The biosynthetic logic for their production is unknown and defies explanation via classical biochemical paradigms. A large subgroup of naphthoquinone-based meroterpenoids exhibits a substitution pattern of the polyketide-derived aromatic core that seemingly contradicts the established reactivity pattern of polyketide phenol nucleophiles and terpene diphosphate electrophiles. We report the discovery of a hitherto unprecedented enzyme-promoted α-hydroxyketone rearrangement catalysed by vanadium-dependent haloperoxidases to account for these discrepancies in the merochlorin and napyradiomycin class of meroterpenoid antibiotics, and we demonstrate that the α-hydroxyketone rearrangement is potentially a conserved biosynthetic reaction in this molecular class. The biosynthetic α-hydroxyketone rearrangement was applied in a concise total synthesis of naphthomevalin, a prominent member of the napyradiomycin meroterpenes, and sheds further light on the mechanism of this unifying enzymatic transformation.

Identification and Characterization of the Pyridomycin Biosynthetic Gene Cluster of Streptomyces pyridomyceticus NRRL B-2517*

PubMed Central

Huang, Tingting; Wang, Yemin; Yin, Jun; Du, Yanhua; Tao, Meifeng; Xu, Jing; Chen, Wenqing; Lin, Shuangjun; Deng, Zixin

2011-01-01

Pyridomycin is a structurally unique antimycobacterial cyclodepsipeptide containing rare 3-(3-pyridyl)-l-alanine and 2-hydroxy-3-methylpent-2-enoic acid moieties. The biosynthetic gene cluster for pyridomycin has been cloned and identified from Streptomyces pyridomyceticus NRRL B-2517. Sequence analysis of a 42.5-kb DNA region revealed 26 putative open reading frames, including two nonribosomal peptide synthetase (NRPS) genes and a polyketide synthase gene. A special feature is the presence of a polyketide synthase-type ketoreductase domain embedded in an NRPS. Furthermore, we showed that PyrA functioned as an NRPS adenylation domain that activates 3-hydroxypicolinic acid and transfers it to a discrete peptidyl carrier protein, PyrU, which functions as a loading module that initiates pyridomycin biosynthesis in vivo and in vitro. PyrA could also activate other aromatic acids, generating three pyridomycin analogues in vivo. PMID:21454714

Engineered fungal polyketide biosynthesis in Pichia pastoris: a potential excellent host for polyketide production

PubMed Central

2013-01-01

Background Polyketides are one of the most important classes of secondary metabolites and usually make good drugs. Currently, heterologous production of fungal polyketides for developing a high potential industrial application system with high production capacity and pharmacutical feasibility was still at its infancy. Pichia pastoris is a highly successful system for the high production of a variety of heterologous proteins. In this work, we aim to develop a P. pastoris based in vivo fungal polyketide production system for first time and evaluate its feasibility for future industrial application. Results A recombinant P. pastoris GS115-NpgA-ATX with Aspergillus nidulans phosphopantetheinyl transferase (PPtase) gene npgA and Aspergillus terrus 6-methylsalicylic acid (6-MSA) synthase (6-MSAS) gene atX was constructed. A specific compound was isolated and idenified as 6-MSA by HPLC, LC-MS and NMR. Transcription of both genes were detected. In 5-L bioreactor, the GS115-NpgA-ATX grew well and produced 6-MSA quickly until reached a high value of 2.2 g/L by methanol induction for 20 hours. Thereafter, the cells turned to death ascribing to high concentration of antimicrobial 6-MSA. The distribution of 6-MSA changed that during early and late induction phase it existed more in supernatant while during intermediate stage it mainly located intracellular. Different from 6-MSA production strain, recombinant M. purpureus pksCT expression strains for citrinin intermediate production, no matter PksCT located in cytoplasm or in peroxisomes, did not produce any specfic compound. However, both npgA and pksCT transcripted effectively in cells and western blot analysis proved the expression of PPtase. Then the PPTase was expressed and purified, marked by fluorescent probes, and reacted with purified ACP domain and its mutant ACPm of PksCT. Fluoresence was only observed in ACP but not ACPm, indicating that the PPTase worked well with ACP to make it bioactive holo-ACP. Thus, some other factors may affect polyketide synthesis that include activities of the individual catalytic domains and release of the product from the synthase of PksCT. Conclusions An efficient P. pastoris expression system of fungal polyketides was successfully constructed. It produced a high production of 6-MSA and holds potential for future industrial application of 6-MSA and other fungal polyketides. PMID:24011431

Engineered fungal polyketide biosynthesis in Pichia pastoris: a potential excellent host for polyketide production.

PubMed

Gao, Limei; Cai, Menghao; Shen, Wei; Xiao, Siwei; Zhou, Xiangshan; Zhang, Yuanxing

2013-09-08

Polyketides are one of the most important classes of secondary metabolites and usually make good drugs. Currently, heterologous production of fungal polyketides for developing a high potential industrial application system with high production capacity and pharmaceutical feasibility was still at its infancy. Pichia pastoris is a highly successful system for the high production of a variety of heterologous proteins. In this work, we aim to develop a P. pastoris based in vivo fungal polyketide production system for first time and evaluate its feasibility for future industrial application. A recombinant P. pastoris GS115-NpgA-ATX with Aspergillus nidulans phosphopantetheinyl transferase (PPtase) gene npgA and Aspergillus terrus 6-methylsalicylic acid (6-MSA) synthase (6-MSAS) gene atX was constructed. A specific compound was isolated and identified as 6-MSA by HPLC, LC-MS and NMR. Transcription of both genes were detected. In 5-L bioreactor, the GS115-NpgA-ATX grew well and produced 6-MSA quickly until reached a high value of 2.2 g/L by methanol induction for 20 hours. Thereafter, the cells turned to death ascribing to high concentration of antimicrobial 6-MSA. The distribution of 6-MSA changed that during early and late induction phase it existed more in supernatant while during intermediate stage it mainly located intracellular. Different from 6-MSA production strain, recombinant M. purpureus pksCT expression strains for citrinin intermediate production, no matter PksCT located in cytoplasm or in peroxisomes, did not produce any specific compound. However, both npgA and pksCT transcripted effectively in cells and western blot analysis proved the expression of PPtase. Then the PPTase was expressed and purified, marked by fluorescent probes, and reacted with purified ACP domain and its mutant ACPm of PksCT. Fluoresence was only observed in ACP but not ACPm, indicating that the PPTase worked well with ACP to make it bioactive holo-ACP. Thus, some other factors may affect polyketide synthesis that include activities of the individual catalytic domains and release of the product from the synthase of PksCT. An efficient P. pastoris expression system of fungal polyketides was successfully constructed. It produced a high production of 6-MSA and holds potential for future industrial application of 6-MSA and other fungal polyketides.

Biosynthesis and regulation of coronatine, a non-host-specific phytotoxin produced by Pseudomonas syringae.

PubMed

Bender, C L; Palmer, D A; Peñaloza-Vázquez, A; Rangaswamy, V; Ullrich, M

1998-01-01

Many P. syringae pathovars are known to produce low-molecular-weight, diffusible toxins in infected host plants. These phytotoxins reproduce some of the symptoms of the relevant bacterial disease and are effective at very low concentrations. Phytotoxins generally enhance the virulence of the P. syringae pathovar which produces them, but are not required for pathogenesis. Genes encoding phytotoxin production have been identified and cloned from several P. syringae pathovars. With the exception of coronatine, toxin biosynthetic gene clusters are generally chromosomally encoded. In several pathovars, the toxin biosynthetic gene cluster also contains a resistance gene which functions to protect the producing strain from the biocidal effects of the toxin. In the case of phaseolotoxin, a resistance gene (argK) has been utilized to engineer phaseolotoxin-resistant tobacco plants. Although P. syringae phytotoxins can induce very similar effects in plants (chlorosis and necrosis), their biosynthesis and mode of action can be quite different. Knowledge of the biosynthetic pathways to these toxins and the cloning of the structural genes for their biosynthesis has relevance to the development of new bioactive compounds with altered specificity. For example, polyketides constitute a huge family of structurally diverse natural products including antibiotics, chemotherapeutic compounds, and antiparasitics. Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by Streptomyces or other actinomycetes. It is also important to note that it is now possible to utilize a genetic rather than synthetic approach to biosynthesize novel polyketides with altered biological properties (Hutchinson and Fujii, 1995; Kao et al., 1994; Donadio et al., 1993; Katz and Donadio, 1993). Most of the reprogramming or engineering of novel polyketides has been done using actinomycete PKSs, but much of this technology could also be applied to polyketides synthesized by Pseudomonas when sufficient sequence information is available. It is important to note that Pseudomonas produces a variety of antimicrobial compounds from the polyketide pathway, including mupirocin (pseudomonic acid) (Feline et al., 1977), pyoluteorin (Cuppels et al., 1986), and 2-4 diacetylphloroglucinol (Phl) (Bangera and Thomashow, 1996). Pseudomonic acid is valued for its pharmaceutical properties as an antibiotic (Aldridge, 1992), whereas pyoluteorin and Phl have antifungal properties (Howell and Stipanovic, 1980; Keel et al., 1992). A thorough understanding of the biosynthetic pathway to polyketide phytotoxins such as coronatine may ultimately lead to the development of novel compounds with altered biological properties. Thus, specific genes in the biosynthetic pathways of P. syringae phytotoxins could be deployed in other systems to develop new compounds with a wide range of activities.

Nocardiopsis species: a potential source of bioactive compounds.

PubMed

Bennur, T; Ravi Kumar, A; Zinjarde, S S; Javdekar, V

2016-01-01

Members of the genus Nocardiopsis are an ecologically versatile and biotechnologically important group of Actinomycetes. Most of the isolates are halotolerant or halophilic and they prevail in soils, marine environments or hypersaline locations. To aid their survival under these conditions, they mainly produce extremozymes, compatible solutes, surfactants and bioactive compounds. The current review details the bioactive compounds obtained for this genus. Important antimicrobial agents obtained from this genus include polyketides, phenzines, quinoline alkaloids, terphenyls, proteins, thiopeptides and amines. Polyketides and peptides displaying potent anticancer activities are also significant. Tumour promoting agents, P-glycoprotein (P-gp) inhibitors, immunomodulators and protein kinase inhibitors are other relevant products obtained from Nocardiopsis species. Structurally, polyketides (synthesized by polyketide synthases) and peptides (made by nonribosomal peptide synthetases or cyclodipeptide synthases) are important compounds. Considered here are also toxins, anti photoaging and adipogenic agents produced by this genus. The gene clusters mediating the synthesis of bioactive compounds have been described. Commercially available products (Apoptolidins and K-252a) derived from this genus have also been described. This review highlights the significance of a single genus in producing an assortment of compounds with varied biological activities. On account of these features, the members of this genus have established a place for themselves and are of considerable value in producing compounds with profound bio-medical applications. © 2015 The Society for Applied Microbiology.

Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

PubMed

Gagne, Steve J; Stout, Jake M; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M; Page, Jonathan E

2012-07-31

Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

Toblerols: Cyclopropanol-Containing Polyketide Modulators of Antibiosis in Methylobacteria.

PubMed

Ueoka, Reiko; Bortfeld-Miller, Miriam; Morinaka, Brandon I; Vorholt, Julia A; Piel, Jörn

2018-01-22

Trans-AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular type I PKSs that generate bioactive polyketides of impressive structural diversity. In this study, we detected, in the genome of several bacteria a cryptic, architecturally unusual trans-AT PKS gene cluster which eluded automated PKS prediction. Genomic mining of one of these strains, the model methylotroph Methylobacterium extorquens AM1, revealed unique epoxide- and cyclopropanol-containing polyketides named toblerols. Relative and absolute stereochemistry were determined by NMR experiments, chemical derivatization, and the comparison of CD data between the derivatized natural product and a synthesized model compound. Biosynthetic data suggest that the cyclopropanol moiety is generated by carbon-carbon shortening of a more extended precursor. Surprisingly, a knock-out strain impaired in polyketide production showed strong inhibitory activity against other methylobacteria in contrast to the wild-type producer. The activity was inhibited by complementation with toblerols, thus suggesting that these compounds modulate an as-yet unknown methylobacterial antibiotic. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus.

PubMed

Wang, Peng; Gao, Xue; Chooi, Yit-Heng; Deng, Zixin; Tang, Yi

2011-08-01

Tetracyclines are clinically important aromatic polyketides whose biosynthesis is catalysed by bacterial type II polyketide synthases (PKSs). Tetracyclines are biosynthesized starting with an amide-containing malonamate starter unit and the resulting C-2 carboxyamide is critical for the antibiotic activities. In this work, we genetically verified that an amidotransferase, OxyD, and a thiolase, OxyP, are involved in the biosynthesis and incorporation of the starter unit. First, two mutations, R248T and D268N, were found to be present in OxyD* encoded in Streptomyces rimosus ATCC 13224, a strain that produces the acetate-primed 2-acetyl-2-decarboxyamido-oxytetracycline (ADOTC) instead of the malonamate-primed oxytetracycline (OTC). Homology modelling suggested that in particular D268N may inactivate OxyD. Complementation of S. rimosus ATCC 13224 with wild-type OxyD restored OTC biosynthesis, thereby confirming the essential role of OxyD in the synthesis of the amide starter unit. Second, using a series of knockout and complementation approaches, we demonstrated that OxyP is most likely involved in maintaining fidelity of the amide-priming process via hydrolysis of the competing acetate priming starter units. While the inactivation of OxyP does not eliminate OTC biosynthesis, the ratio of acetate-primed ADOTC to malonamate-primed OTC is significantly increased. This suggests that OxyP plays an ancillary role in OTC biosynthesis and is important for minimizing the levels of ADOTC, a shunt product that has much weaker antibiotic activities than OTC.

Functional Analysis of the Polyketide Synthase Genes in the Filamentous Fungus Gibberella zeae (Anamorph Fusarium graminearum)

PubMed Central

Gaffoor, Iffa; Brown, Daren W.; Plattner, Ron; Proctor, Robert H.; Qi, Weihong; Trail, Frances

2005-01-01

Polyketides are a class of secondary metabolites that exhibit a vast diversity of form and function. In fungi, these compounds are produced by large, multidomain enzymes classified as type I polyketide synthases (PKSs). In this study we identified and functionally disrupted 15 PKS genes from the genome of the filamentous fungus Gibberella zeae. Five of these genes are responsible for producing the mycotoxins zearalenone, aurofusarin, and fusarin C and the black perithecial pigment. A comprehensive expression analysis of the 15 genes revealed diverse expression patterns during grain colonization, plant colonization, sexual development, and mycelial growth. Expression of one of the PKS genes was not detected under any of 18 conditions tested. This is the first study to genetically characterize a complete set of PKS genes from a single organism. PMID:16278459

HSQC-TOCSY Fingerprinting for Prioritization of Polyketide- and Peptide-Producing Microbial Isolates.

PubMed

Buedenbender, Larissa; Habener, Leesa J; Grkovic, Tanja; Kurtböke, D İpek; Duffy, Sandra; Avery, Vicky M; Carroll, Anthony R

2018-04-27

Microbial products are a promising source for drug leads as a result of their unique structural diversity. However, reisolation of already known natural products significantly hampers the discovery process, and it is therefore important to incorporate effective microbial isolate selection and dereplication protocols early in microbial natural product studies. We have developed a systematic approach for prioritization of microbial isolates for natural product discovery based on heteronuclear single-quantum correlation-total correlation spectroscopy (HSQC-TOCSY) nuclear magnetic resonance profiles in combination with antiplasmodial activity of extracts. The HSQC-TOCSY experiments allowed for unfractionated microbial extracts containing polyketide and peptidic natural products to be rapidly identified. Here, we highlight how this approach was used to prioritize extracts derived from a library of 119 ascidian-associated actinomycetes that possess a higher potential to produce bioactive polyketides and peptides.

FUBT, a putative MFS transporter, promotes secretion of fusaric acid in the cotton pathogen Fusarium oxysporum f.sp. vasinfectum

USDA-ARS?s Scientific Manuscript database

Fusaric acid (FA), a phytotoxic polyketide produced by Fusarium oxysporum f. sp. vasinfectum (FOV), has been shown to be associated with disease symptoms on cotton. A gene located upstream of the polyketide synthase gene responsible for the biosynthesis of FA is predicted to encode a member of the ...

Producing biofuels using polyketide synthases

DOEpatents

Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

2013-04-16

The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

A novel polyketide biosynthesis gene cluster is involved in fruiting body morphogenesis in the filamentous fungi Sordaria macrospora and Neurospora crassa.

PubMed

Nowrousian, Minou

2009-04-01

During fungal fruiting body development, hyphae aggregate to form multicellular structures that protect and disperse the sexual spores. Analysis of microarray data revealed a gene cluster strongly upregulated during fruiting body development in the ascomycete Sordaria macrospora. Real time PCR analysis showed that the genes from the orthologous cluster in Neurospora crassa are also upregulated during development. The cluster encodes putative polyketide biosynthesis enzymes, including a reducing polyketide synthase. Analysis of knockout strains of a predicted dehydrogenase gene from the cluster showed that mutants in N. crassa and S. macrospora are delayed in fruiting body formation. In addition to the upregulated cluster, the N. crassa genome comprises another cluster containing a polyketide synthase gene, and five additional reducing polyketide synthase (rpks) genes that are not part of clusters. To study the role of these genes in sexual development, expression of the predicted rpks genes in S. macrospora (five genes) and N. crassa (six genes) was analyzed; all but one are upregulated during sexual development. Analysis of knockout strains for the N. crassa rpks genes showed that one of them is essential for fruiting body formation. These data indicate that polyketides produced by RPKSs are involved in sexual development in filamentous ascomycetes.

Characterization of the Biosynthetic Genes for 10,11-Dehydrocurvularin, a Heat Shock Response-Modulating Anticancer Fungal Polyketide from Aspergillus terreus

PubMed Central

Xu, Yuquan; Espinosa-Artiles, Patricia; Schubert, Vivien; Xu, Ya-ming; Zhang, Wei; Lin, Min; Gunatilaka, A. A. Leslie; Süssmuth, Roderich

2013-01-01

10,11-Dehydrocurvularin is a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities. It features a dihydroxyphenylacetic acid lactone polyketide framework with structural similarities to resorcylic acid lactones like radicicol or zearalenone. A genomic locus was identified from the dehydrocurvularin producer strain Aspergillus terreus AH-02-30-F7 to reveal genes encoding a pair of iterative polyketide synthases (A. terreus CURS1 [AtCURS1] and AtCURS2) that are predicted to collaborate in the biosynthesis of 10,11-dehydrocurvularin. Additional genes in this locus encode putative proteins that may be involved in the export of the compound from the cell and in the transcriptional regulation of the cluster. 10,11-Dehydrocurvularin biosynthesis was reconstituted in Saccharomyces cerevisiae by heterologous expression of the polyketide synthases. Bioinformatic analysis of the highly reducing polyketide synthase AtCURS1 and the nonreducing polyketide synthase AtCURS2 highlights crucial biosynthetic programming differences compared to similar synthases involved in resorcylic acid lactone biosynthesis. These differences lead to the synthesis of a predicted tetraketide starter unit that forms part of the 12-membered lactone ring of dehydrocurvularin, as opposed to the penta- or hexaketide starters in the 14-membered rings of resorcylic acid lactones. Tetraketide N-acetylcysteamine thioester analogues of the starter unit were shown to support the biosynthesis of dehydrocurvularin and its analogues, with yeast expressing AtCURS2 alone. Differential programming of the product template domain of the nonreducing polyketide synthase AtCURS2 results in an aldol condensation with a different regiospecificity than that of resorcylic acid lactones, yielding the dihydroxyphenylacetic acid scaffold characterized by an S-type cyclization pattern atypical for fungal polyketides. PMID:23335766

United States Department of Agriculture-Agricultural Research Service studies on polyketide toxins of Fusarium oxysporum f sp vasinfectum: potential targets for disease control.

PubMed

Bell, Alois A; Wheeler, Michael H; Liu, Jinggao; Stipanovic, Robert D; Puckhaber, Lorraine S; Orta, Heather

2003-01-01

A group of 133 isolates of the cotton wilt pathogen Fusarium oxysporum Schlecht f sp vasinfectum (Atk) Sny & Hans, representing five races and 20 vegetative compatibility groups within race 1 were used to determine the identity, biosynthetic regulation and taxonomic distribution of polyketide toxins produced by this pathogen. All isolates of F oxysporum f sp vasinfectum produced and secreted the nonaketide naphthazarin quinones, bikaverin and norbikaverin. Most isolates of race 1 (previously denoted as races 1, 2 and 6; and also called race A) also synthesized the heptaketide naphthoquinones, nectriafurone, anhydrofusarubin lactol and 5-O-methyljavanicin. Nine avirulent isolates of F oxysporum from Upland cotton roots, three isolates of race 3 of F oxysporum f sp vasinfectum, and four isolates of F oxysporum f sp vasinfectum from Australia, all of which previously failed to cause disease of Upland cotton (Gossypium hirsutum L) in stem-puncture assays, also failed to synthesize or secrete more than trace amounts of the heptaketide compounds. These results indicate that the heptaketides may have a unique role in the virulence of race 1 to Upland cotton. The synthesis of all polyketide toxins by ATCC isolate 24908 of F oxysporum f sp vasinfectum was regulated by pH, carbon/nitrogen ratios, and availability of calcium in media. Synthesis was greatest below pH 7.0 and increased progressively as carbon/nitrogen ratios were increased by decreasing the amounts of nitrogen added to media. The nonaketides were the major polyketides accumulated in synthetic media at pH 4.5 and below, whereas the heptaketides were predominant at pH 5.0 and above. The heptaketides were the major polyketides formed when 10 F oxysporum f sp vasinfectum race 1 isolates were grown on sterilized stems of Fusarium wilt-susceptible cotton cultivars, but these compounds were not produced on sorghum grain cultures. Both groups of polyketide toxins were apparently secreted by F oxysporum f sp vasinfectum, since half of the toxin in 2-day-old shake culture was present in the supernatant. Secretion was enhanced by calcium. Glutamine and glutamic acid inhibited both nonaketide and heptaketide syntheses, even at low nitrogen

The polyketide synthase gene pks4 is essential for sexual development and regulates fruiting body morphology in Sordaria macrospora.

PubMed

Schindler, Daniel; Nowrousian, Minou

2014-07-01

Filamentous ascomycetes have long been known as producers of a variety of secondary metabolites, many of which have toxic effects on other organisms. However, the role of these metabolites in the biology of the fungi that produce them remains in most cases enigmatic. A major group of fungal secondary metabolites are polyketides. They are chemically diverse, but have in common that their chemical scaffolds are synthesized by polyketide synthases (PKSs). In a previous study, we analyzed development-dependent expression of pks genes in the filamentous ascomycete Sordaria macrospora. Here, we show that a deletion mutant of the pks4 gene is sterile, producing only protoperithecia but no mature perithecia, whereas overexpression of pks4 leads to enlarged, malformed fruiting bodies. Thus, correct expression levels of pks4 are essential for wild type-like perithecia formation. The predicted PKS4 protein has a domain structure that is similar to homologs in other fungi, but conserved residues of a methyl transferase domain present in other fungi are mutated in PKS4. Expression of several developmental genes is misregulated in the pks4 mutant. Surprisingly, the development-associated app gene is not downregulated in the mutant, in contrast to all other previously studied mutants with a block at the protoperithecial stage. Our data show that the polyketide synthase gene pks4 is essential for sexual development and plays a role in regulating fruiting body morphology. Copyright © 2014 Elsevier Inc. All rights reserved.

Novel Artificial Natural Products Against Breast Cancer Through Combinatorial Biosynthesis

DTIC Science & Technology

2002-07-01

compounds normally produced by a certain strain. Our investigations on the discovery of novel natural metabolites using type II polyketide synthase ...limitations, shall be included on any reproduction hereof which includes any part of the portions subject to such limitations. THIS TECHNICAL REPORT HAS... polyketides remain the central group of natural products in this research area, since this class of natural products form one of the largest and most

Allergens/Antigens, toxins and polyketides of important Aspergillus species.

PubMed

Bhetariya, Preetida J; Madan, Taruna; Basir, Seemi Farhat; Varma, Anupam; Usha, Sarma P

2011-04-01

The medical, agricultural and biotechnological importance of the primitive eukaryotic microorganisms, the Fungi was recognized way back in 1920. Among various groups of fungi, the Aspergillus species are studied in great detail using advances in genomics and proteomics to unravel biological and molecular mechanisms in these fungi. Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, Aspergillus nidulans and Aspergillus terreus are some of the important species relevant to human, agricultural and biotechnological applications. The potential of Aspergillus species to produce highly diversified complex biomolecules such as multifunctional proteins (allergens, antigens, enzymes) and polyketides is fascinating and demands greater insight into the understanding of these fungal species for application to human health. Recently a regulator gene for secondary metabolites, LaeA has been identified. Gene mining based on LaeA has facilitated new metabolites with antimicrobial activity such as emericellamides and antitumor activity such as terrequinone A from A. nidulans. Immunoproteomic approach was reported for identification of few novel allergens for A. fumigatus. In this context, the review is focused on recent developments in allergens, antigens, structural and functional diversity of the polyketide synthases that produce polyketides of pharmaceutical and biological importance. Possible antifungal drug targets for development of effective antifungal drugs and new strategies for development of molecular diagnostics are considered.

Divergent Mechanistic Routes for the Formation of gem-Dimethyl Groups in the Biosynthesis of Complex Polyketides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poust, S; Phelan, RM; Deng, K

The gem-dimethyl groups in polyketide-derived natural products add steric bulk and, accordingly, lend increased stability to medicinal compounds, however, our ability to rationally incorporate this functional group in modified natural products is limited. In order to characterize the mechanism of gem-dimethyl group formation, with a goal toward engineering of novel compounds containing this moiety, the gem-dimethyl group producing polyketide synthase (PKS) modules of yersiniabactin and epothilone were characterized using mass spectrometry. The work demonstrated, contrary to the canonical understanding of reaction order in PKSs, that methylation can precede condensation in gem-dimethyl group producing PKS modules. Experiments showed that both PKSsmore » are able to use dimethylmalonyl acyl carrier protein (ACP) as an extender unit. Interestingly, for epothilone module8, use of dimethylmalonyl-ACP appeared to be the sole route to form a gem-dimethylated product, while the yersiniabactin PKS could methylate before or after ketosynthase condensation.« less

A dual role for a polyketide synthase in dynemicin enediyne and anthraquinone biosynthesis

NASA Astrophysics Data System (ADS)

Cohen, Douglas R.; Townsend, Craig A.

2018-02-01

Dynemicin A is a member of a subfamily of enediyne antitumour antibiotics characterized by a 10-membered carbocycle fused to an anthraquinone, both of polyketide origin. Sequencing of the dynemicin biosynthetic gene cluster in Micromonospora chersina previously identified an enediyne polyketide synthase (PKS), but no anthraquinone PKS, suggesting gene(s) for biosynthesis of the latter were distant from the core dynemicin cluster. To identify these gene(s), we sequenced and analysed the genome of M. chersina. Sequencing produced a short list of putative PKS candidates, yet CRISPR-Cas9 mutants of each locus retained dynemicin production. Subsequently, deletion of two cytochromes P450 in the dynemicin cluster suggested that the dynemicin enediyne PKS, DynE8, may biosynthesize the anthraquinone. Together with 18O-labelling studies, we now present evidence that DynE8 produces the core scaffolds of both the enediyne and anthraquinone, and provide a working model to account for their formation from the programmed octaketide of the enediyne PKS.

Improved pestalotiollide B production by deleting competing polyketide synthase genes in Pestalotiopsis microspora.

PubMed

Chen, Longfei; Li, Yingying; Zhang, Qian; Wang, Dan; Akhberdi, Oren; Wei, Dongsheng; Pan, Jiao; Zhu, Xudong

2017-02-01

Pestalotiollide B, an analog of dibenzodioxocinones which are inhibitors of cholesterol ester transfer proteins, is produced by Pestalotiopsis microspora NK17. To increase the production of pestalotiollide B, we attempted to eliminate competing polyketide products by deleting the genes responsible for their biosynthesis. We successfully deleted 41 out of 48 putative polyketide synthases (PKSs) in the genome of NK17. Nine of the 41 PKS deleted strains had significant increased production of pestalotiollide B (P < 0.05). For instance, deletion of pks35, led to an increase of pestalotiollide B by 887%. We inferred that these nine PKSs possibly lead to branch pathways that compete for precursors with pestalotiollide B, or that convert the product. Deletion of some other PKS genes such as pks8 led to a significant decrease of pestalotiollide B, suggesting they are responsible for its biosynthesis. Our data demonstrated that improvement of pestalotiollide B production can be achieved by eliminating competing polyketides.

Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hagen, Andrew; Poust, Sean; Rond, Tristan de

2015-10-26

Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design–build–test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS’ first extensionmore » module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to “debug” PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.« less

Conversion of the high-yield salinomycin producer Streptomyces albus BK3-25 into a surrogate host for polyketide production.

PubMed

Zhang, Xiaojie; Lu, Chenyang; Bai, Linquan

2017-09-01

An ideal surrogate host for heterologous production of various natural products is expected to have efficient nutrient utilization, fast growth, abundant precursors and energy supply, and a pronounced gene expression. Streptomyces albus BK3-25 is a high-yield industrial strain producing type-I polyketide salinomycin, with a unique ability of bean oil utilization. Its potential of being a surrogate host for heterologous production of PKS was engineered and evaluated herein. Firstly, introduction of a three-gene cassette for the biosynthesis of ethylmalonyl-CoA resulted in accumulation of ethylmalonyl-CoA precursor and salinomycin, and subsequent deletion of the salinomycin biosynthetic gene cluster resulted in a host with rich supplies of common polyketide precursors, including malonyl-CoA, methylmalonyl-CoA, and ethylmalonyl-CoA. Secondly, the energy and reducing force were measured, and the improved accumulation of ATP and NADPH was observed in the mutant. Furthermore, the strength of a series of selected endogenous promoters based on microarray data was assessed at different growth phases, and a strong constitutive promoter was identified, providing a useful tool for further engineered gene expression. Finally, the potential of the BK3-25 derived host ZXJ-6 was evaluated with the introduction of the actinorhodin biosynthetic gene cluster from Streptomyces coelicolor, and the heterologous production of actinorhodin was obtained. This work clearly indicated the potential of the high-yield salinomycin producer as a surrogate host for heterologous production of polyketides, although more genetic manipulation should be conducted to streamline its performance.

Phylogenomic and Domain Analysis of Iterative Polyketide Synthases in Aspergillus Species

PubMed Central

Lin, Shu-Hsi; Yoshimoto, Miwa; Lyu, Ping-Chiang; Tang, Chuan-Yi; Arita, Masanori

2012-01-01

Aspergillus species are industrially and agriculturally important as fermentors and as producers of various secondary metabolites. Among them, fungal polyketides such as lovastatin and melanin are considered a gold mine for bioactive compounds. We used a phylogenomic approach to investigate the distribution of iterative polyketide synthases (PKS) in eight sequenced Aspergilli and classified over 250 fungal genes. Their genealogy by the conserved ketosynthase (KS) domain revealed three large groups of nonreducing PKS, one group inside bacterial PKS, and more than 9 small groups of reducing PKS. Polyphyly of nonribosomal peptide synthase (NRPS)-PKS genes raised questions regarding the recruitment of the elegant conjugation machinery. High rates of gene duplication and divergence were frequent. All data are accessible through our web database at http://metabolomics.jp/wiki/Category:PK. PMID:22844193

Engineering Biosynthesis of Non-ribosomal Peptides and Polyketides by Directed Evolution.

PubMed

Rui, Zhe; Zhang, Wenjun

2016-01-01

Non-ribosomal peptides (NRPs) and polyketides (PKs) play key roles in pharmaceutical industry due to their promising biological activities. The structural complexity of NRPs and PKs, however, creates significant synthetic challenges for producing these natural products and their analogues by purely chemical means. Alternatively, difficult syntheses can be achieved by using biosynthetic enzymes with improved efficiency and altered selectivity that are acquired from directed evolution. Key to the successful directed evolution is the methodology of screening/selection. This review summarizes the screening/selection strategies that have been employed to improve or modify the functions of non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), in the hope of triggering the wide adoption of the directed evolution approaches in the engineered biosynthesis of NRPs and PKs for drug discovery.

Comparative Genomics and an Insect Model Rapidly Identify Novel Virulence Genes of Burkholderia mallei

DTIC Science & Technology

2008-04-01

IID on A pril 23, 2008 jb.asm .org D ow nloaded from metabolite-producing clusters encoding nonribosomal peptide or polyketide synthetases...BMA1848) encod- ing a subunit of acetolactate synthase III. The resultant mutant was not able to grow on minimal glucose medium and, similar to what has...caused by the wild type. BMAA1204 is a 4,200-residue CDS annotated as encoding a putative polyketide synthase (PKS) in COG family 0332. PKSs are

Identification and regulation of fusA, the polyketide synthase gene responsible for fusarin production in Fusarium fujikuroi.

PubMed

Díaz-Sánchez, Violeta; Avalos, Javier; Limón, M Carmen

2012-10-01

Fusarins are a class of mycotoxins of the polyketide family produced by different Fusarium species, including the gibberellin-producing fungus Fusarium fujikuroi. Based on sequence comparisons between polyketide synthase (PKS) enzymes for fusarin production in other Fusarium strains, we have identified the F. fujikuroi orthologue, called fusA. The participation of fusA in fusarin biosynthesis was demonstrated by targeted mutagenesis. Fusarin production is transiently stimulated by nitrogen availability in this fungus, a regulation paralleled by the fusA mRNA levels in the cell. Illumination of the cultures results in a reduction of the fusarin content, an effect partially explained by a high sensitivity of these compounds to light. Mutants of the fusA gene exhibit no external phenotypic alterations, including morphology and conidiation, except for a lack of the characteristic yellow and/or orange pigmentation of fusarins. Moreover, the fusA mutants are less efficient than the wild type at degrading cellophane on agar cultures, a trait associated with pathogenesis functions in Fusarium oxysporum. The fusA mutants, however, are not affected in their capacities to grow on plant tissues.

Ketide Synthase (KS) Domain Prediction and Analysis of Iterative Type II PKS Gene in Marine Sponge-Associated Actinobacteria Producing Biosurfactants and Antimicrobial Agents

PubMed Central

Selvin, Joseph; Sathiyanarayanan, Ganesan; Lipton, Anuj N.; Al-Dhabi, Naif Abdullah; Valan Arasu, Mariadhas; Kiran, George S.

2016-01-01

The important biological macromolecules, such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS) genes was explored. A unique feature of type II PKS genes is their high amino acid (AA) sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of various domains. Therefore, deciphering the order of AA sequence encoded by PKS domains tailored the chemical structure of polyketide analogs still remains a great challenge. The present work deals with an in vitro and in silico analysis of PKS type II genes from five actinobacterial species to correlate KS domain architecture and structural features. Our present analysis reveals the unique protein domain organization of iterative type II PKS and KS domain of marine actinobacteria. The findings of this study would have implications in metabolic pathway reconstruction and design of semi-synthetic genomes to achieve rational design of novel natural products. PMID:26903957

Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics.

PubMed

Gold, Nicholas D; Gowen, Christopher M; Lussier, Francois-Xavier; Cautha, Sarat C; Mahadevan, Radhakrishnan; Martin, Vincent J J

2015-05-28

L-tyrosine is a common precursor for a wide range of valuable secondary metabolites, including benzylisoquinoline alkaloids (BIAs) and many polyketides. An industrially tractable yeast strain optimized for production of L-tyrosine could serve as a platform for the development of BIA and polyketide cell factories. This study applied a targeted metabolomics approach to evaluate metabolic engineering strategies to increase the availability of intracellular L-tyrosine in the yeast Saccharomyces cerevisiae CEN.PK. Our engineering strategies combined localized pathway engineering with global engineering of central metabolism, facilitated by genome-scale steady-state modelling. Addition of a tyrosine feedback resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase Aro4 from S. cerevisiae was combined with overexpression of either a tyrosine feedback resistant yeast chorismate mutase Aro7, the native pentafunctional arom protein Aro1, native prephenate dehydrogenase Tyr1 or cyclohexadienyl dehydrogenase TyrC from Zymomonas mobilis. Loss of aromatic carbon was limited by eliminating phenylpyruvate decarboxylase Aro10. The TAL gene from Rhodobacter sphaeroides was used to produce coumarate as a simple test case of a heterologous by-product of tyrosine. Additionally, multiple strategies for engineering global metabolism to promote tyrosine production were evaluated using metabolic modelling. The T21E mutant of pyruvate kinase Cdc19 was hypothesized to slow the conversion of phosphoenolpyruvate to pyruvate and accumulate the former as precursor to the shikimate pathway. The ZWF1 gene coding for glucose-6-phosphate dehydrogenase was deleted to create an NADPH deficiency designed to force the cell to couple its growth to tyrosine production via overexpressed NADP(+)-dependent prephenate dehydrogenase Tyr1. Our engineered Zwf1(-) strain expressing TYRC ARO4(FBR) and grown in the presence of methionine achieved an intracellular L-tyrosine accumulation up to 520 μmol/g DCW or 192 mM in the cytosol, but sustained flux through this pathway was found to depend on the complete elimination of feedback inhibition and degradation pathways. Our targeted metabolomics approach confirmed a likely regulatory site at DAHP synthase and identified another possible cofactor limitation at prephenate dehydrogenase. Additionally, the genome-scale metabolic model identified design strategies that have the potential to improve availability of erythrose 4-phosphate for DAHP synthase and cofactor availability for prephenate dehydrogenase. We evaluated these strategies and provide recommendations for further improvement of aromatic amino acid biosynthesis in S. cerevisiae.

Insights into natural products biosynthesis from analysis of 490 polyketide synthases from Fusarium.

PubMed

Brown, Daren W; Proctor, Robert H

2016-04-01

Species of the fungus Fusarium collectively cause disease on almost all crop plants and produce numerous natural products (NPs), including some of the mycotoxins of greatest concern to agriculture. Many Fusarium NPs are derived from polyketide synthases (PKSs), large multi-domain enzymes that catalyze sequential condensation of simple carboxylic acids to form polyketides. To gain insight into the biosynthesis of polyketide-derived NPs in Fusarium, we retrieved 488 PKS gene sequences from genome sequences of 31 species of the fungus. In addition to these apparently functional PKS genes, the genomes collectively included 81 pseudogenized PKS genes. Phylogenetic analysis resolved the PKS genes into 67 clades, and based on multiple lines of evidence, we propose that homologs in each clade are responsible for synthesis of a polyketide that is distinct from those synthesized by PKSs in other clades. The presence and absence of PKS genes among the species examined indicated marked differences in distribution of PKS homologs. Comparisons of Fusarium PKS genes and genes flanking them to those from other Ascomycetes provided evidence that Fusarium has the genetic potential to synthesize multiple NPs that are the same or similar to those reported in other fungi, but that have not yet been reported in Fusarium. The results also highlight ways in which such analyses can help guide identification of novel Fusarium NPs and differences in NP biosynthetic capabilities that exist among fungi. Published by Elsevier Inc.

Evolutionary distinctiveness of fatty acid and polyketide synthesis in eukaryotes

PubMed Central

Kohli, Gurjeet S; John, Uwe; Van Dolah, Frances M; Murray, Shauna A

2016-01-01

Fatty acids, which are essential cell membrane constituents and fuel storage molecules, are thought to share a common evolutionary origin with polyketide toxins in eukaryotes. While fatty acids are primary metabolic products, polyketide toxins are secondary metabolites that are involved in ecologically relevant processes, such as chemical defence, and produce the adverse effects of harmful algal blooms. Selection pressures on such compounds may be different, resulting in differing evolutionary histories. Surprisingly, some studies of dinoflagellates have suggested that the same enzymes may catalyse these processes. Here we show the presence and evolutionary distinctiveness of genes encoding six key enzymes essential for fatty acid production in 13 eukaryotic lineages for which no previous sequence data were available (alveolates: dinoflagellates, Vitrella, Chromera; stramenopiles: bolidophytes, chrysophytes, pelagophytes, raphidophytes, dictyochophytes, pinguiophytes, xanthophytes; Rhizaria: chlorarachniophytes, haplosporida; euglenids) and 8 other lineages (apicomplexans, bacillariophytes, synurophytes, cryptophytes, haptophytes, chlorophyceans, prasinophytes, trebouxiophytes). The phylogeny of fatty acid synthase genes reflects the evolutionary history of the organism, indicating selection to maintain conserved functionality. In contrast, polyketide synthase gene families are highly expanded in dinoflagellates and haptophytes, suggesting relaxed constraints in their evolutionary history, while completely absent from some protist lineages. This demonstrates a vast potential for the production of bioactive polyketide compounds in some lineages of microbial eukaryotes, indicating that the evolution of these compounds may have played an important role in their ecological success. PMID:26784357

Genomic and transcriptomic analyses reveal differential regulation of diverse terpenoid and polyketides secondary metabolites in Hericium erinaceus.

PubMed

Chen, Juan; Zeng, Xu; Yang, Yan Long; Xing, Yong Mei; Zhang, Qi; Li, Jia Mei; Ma, Ke; Liu, Hong Wei; Guo, Shun Xing

2017-08-31

The lion's mane mushroom Hericium erinaceus is a famous traditional medicinal fungus credited with anti-dementia activity and a producer of cyathane diterpenoid natural products (erinacines) useful against nervous system diseases. To date, few studies have explored the biosynthesis of these compounds, although their chemical synthesis is known. Here, we report the first genome and tanscriptome sequence of the medicinal fungus H. erinaceus. The size of the genome is 39.35 Mb, containing 9895 gene models. The genome of H. erinaceus reveals diverse enzymes and a large family of cytochrome P450 (CYP) proteins involved in the biosynthesis of terpenoid backbones, diterpenoids, sesquiterpenes and polyketides. Three gene clusters related to terpene biosynthesis and one gene cluster for polyketides biosynthesis (PKS) were predicted. Genes involved in terpenoid biosynthesis were generally upregulated in mycelia, while the PKS gene was upregulated in the fruiting body. Comparative genome analysis of 42 fungal species of Basidiomycota revealed that most edible and medicinal mushroom show many more gene clusters involved in terpenoid and polyketide biosynthesis compared to the pathogenic fungi. None of the gene clusters for terpenoid or polyketide biosynthesis were predicted in the poisonous mushroom Amanita muscaria. Our findings may facilitate future discovery and biosynthesis of bioactive secondary metabolites from H. erinaceus and provide fundamental information for exploring the secondary metabolites in other Basidiomycetes.

Asperpyrone-Type Bis-Naphtho-γ-Pyrones with COX-2-Inhibitory Activities from Marine-Derived Fungus Aspergillus niger.

PubMed

Fang, Wei; Lin, Xiuping; Wang, Jianjiao; Liu, Yonghong; Tao, Huaming; Zhou, Xuefeng

2016-07-20

Bis-naphtho-γ-pyrones (BNPs) are an important group of aromatic polyketides derived from fungi, and asperpyrone-type BNPs are produced primarily by Aspergillus species. The fungal strain Aspergillus niger SCSIO Jcsw6F30, isolated from a marine alga, Sargassum sp., and identified according to its morphological traits and the internal transcribed spacer (ITS) region sequence, was studied for BNPs secondary metabolisms. After HPLC/MS analysis of crude extract of the fermentation broth, 11 asperpyrone-type BNPs were obtained directly and quickly by chromatographic separation in the extract, and those isolated asperpyrone-type BNPs were structurally identified by NMR and MS analyses. All of the BNPs showed weak cytotoxicities against 10 human tumor cells (IC50 > 30 μM). However, three of them, aurasperone F (3), aurasperone C (6) and asperpyrone A (8), exhibited obvious COX-2-inhibitory activities, with the IC50 values being 11.1, 4.2, and 6.4 μM, respectively. This is the first time the COX-2-inhibitory activities of BNPs have been reported.

Bioactive compounds synthesized by non-ribosomal peptide synthetases and type-I polyketide synthases discovered through genome-mining and metagenomics.

PubMed

Nikolouli, Katerina; Mossialos, Dimitris

2012-08-01

Non-ribosomal peptide synthetases (NRPS) and type-I polyketide synthases (PKS-I) are multimodular enzymes involved in biosynthesis of oligopeptide and polyketide secondary metabolites produced by microorganisms such as bacteria and fungi. New findings regarding the mechanisms underlying NRPS and PKS-I evolution illustrate how microorganisms expand their metabolic potential. During the last decade rapid development of bioinformatics tools as well as improved sequencing and annotation of microbial genomes led to discovery of novel bioactive compounds synthesized by NRPS and PKS-I through genome-mining. Taking advantage of these technological developments metagenomics is a fast growing research field which directly studies microbial genomes or specific gene groups and their products. Discovery of novel bioactive compounds synthesized by NRPS and PKS-I will certainly be accelerated through metagenomics, allowing the exploitation of so far untapped microbial resources in biotechnology and medicine.

Role of Modular Polyketide Synthases in the Production of Polyether Ladder Compounds in Ciguatoxin-Producing Gambierdiscus polynesiensis and G. excentricus (Dinophyceae).

PubMed

Kohli, Gurjeet S; Campbell, Katrina; John, Uwe; Smith, Kirsty F; Fraga, Santiago; Rhodes, Lesley L; Murray, Shauna A

2017-09-01

Gambierdiscus, a benthic dinoflagellate, produces ciguatoxins that cause the human illness Ciguatera. Ciguatoxins are polyether ladder compounds that have a polyketide origin, indicating that polyketide synthases (PKS) are involved in their production. We sequenced transcriptomes of Gambierdiscus excentricus and Gambierdiscus polynesiensis and found 264 contigs encoding single domain ketoacyl synthases (KS; G. excentricus: 106, G. polynesiensis: 143) and ketoreductases (KR; G. excentricus: 7, G. polynesiensis: 8) with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 24 contigs (G. excentricus: 3, G. polynesiensis: 21) encoding multiple PKS domains (forming typical type I PKSs modules) were found. The proposed structure produced by one of these megasynthases resembles a partial carbon backbone of a polyether ladder compound. Seventeen contigs encoding single domain KS, KR, s-malonyltransacylase, dehydratase and enoyl reductase with sequence similarity to type II fatty acid synthases (FAS) in plants were found. Type I PKS and type II FAS genes were distinguished based on the arrangement of domains on the contigs and their sequence similarity and phylogenetic clustering with known PKS/FAS genes in other organisms. This differentiation of PKS and FAS pathways in Gambierdiscus is important, as it will facilitate approaches to investigating toxin biosynthesis pathways in dinoflagellates. © 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.

Brocaenols A-C: novel polyketides from a marine derived Penicillium brocae.

PubMed

Bugni, Tim S; Bernan, Valerie S; Greenstein, Michael; Janso, Jeffrey E; Maiese, William M; Mayne, Charles L; Ireland, Chris M

2003-03-07

Chemical investigation of a Penicillium brocae, obtained from a tissue sample of a Fijian Zyzyya sp. sponge, yielded two known diketopiperazines and three novel cytotoxic polyketides, brocaenols A-C. The brocaenols contain an unusual enolized oxepine lactone ring system that to the best of our knowledge is unprecedented in the literature. The structures were elucidated by using 2D-NMR methods including an INADEQUATE experiment. The absolute stereochemistry of brocaenol A was established by using a modified Mosher method. The taxonomy of the producing fungus was elucidated by using both morphological and rDNA sequence analysis.

Trichothecenes and aspinolides produced by Trichoderma arundinaceum regulate expression of Botrytis cinerea genes involved in virulence and growth

USDA-ARS?s Scientific Manuscript database

Trichoderma arundinaceum (Ta37) and Botrytis cinerea (B05.10) produce the sesquiterpenoids harzianum A (HA) and botrydial (BOT), respectively. Ta'Tri5, an HA non-producer mutant, produces high levels of the polyketide compounds aspinolides (Asp) B and C. We analyzed the role of HA and Asp in the B. ...

Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites.

PubMed

Omura, S; Ikeda, H; Ishikawa, J; Hanamoto, A; Takahashi, C; Shinose, M; Takahashi, Y; Horikawa, H; Nakazawa, H; Osonoe, T; Kikuchi, H; Shiba, T; Sakaki, Y; Hattori, M

2001-10-09

Streptomyces avermitilis is a soil bacterium that carries out not only a complex morphological differentiation but also the production of secondary metabolites, one of which, avermectin, is commercially important in human and veterinary medicine. The major interest in this genus Streptomyces is the diversity of its production of secondary metabolites as an industrial microorganism. A major factor in its prominence as a producer of the variety of secondary metabolites is its possession of several metabolic pathways for biosynthesis. Here we report sequence analysis of S. avermitilis, covering 99% of its genome. At least 8.7 million base pairs exist in the linear chromosome; this is the largest bacterial genome sequence, and it provides insights into the intrinsic diversity of the production of the secondary metabolites of Streptomyces. Twenty-five kinds of secondary metabolite gene clusters were found in the genome of S. avermitilis. Four of them are concerned with the biosyntheses of melanin pigments, in which two clusters encode tyrosinase and its cofactor, another two encode an ochronotic pigment derived from homogentiginic acid, and another polyketide-derived melanin. The gene clusters for carotenoid and siderophore biosyntheses are composed of seven and five genes, respectively. There are eight kinds of gene clusters for type-I polyketide compound biosyntheses, and two clusters are involved in the biosyntheses of type-II polyketide-derived compounds. Furthermore, a polyketide synthase that resembles phloroglucinol synthase was detected. Eight clusters are involved in the biosyntheses of peptide compounds that are synthesized by nonribosomal peptide synthetases. These secondary metabolite clusters are widely located in the genome but half of them are near both ends of the genome. The total length of these clusters occupies about 6.4% of the genome.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zargar, Amin; Bailey, Constance B.; Haushalter, Robert W.

Advances in retooling microorganisms have enabled bioproduction of ‘drop-in’ biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gasturbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), ‘drop-in’ biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical propertiesmore » (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel.« less

Total Biosynthesis and Diverse Applications of the Nonribosomal Peptide-Polyketide Siderophore Yersiniabactin

PubMed Central

Ahmadi, Mahmoud Kamal; Fawaz, Samar; Jones, Charles H.; Zhang, Guojian

2015-01-01

Yersiniabactin (Ybt) is a mixed nonribosomal peptide-polyketide natural product natively produced by the pathogen Yersinia pestis. The compound enables iron scavenging capabilities upon host infection and is biosynthesized by a nonribosomal peptide synthetase featuring a polyketide synthase module. This pathway has been engineered for expression and biosynthesis using Escherichia coli as a heterologous host. In the current work, the biosynthetic process for Ybt formation was improved through the incorporation of a dedicated step to eliminate the need for exogenous salicylate provision. When this improvement was made, the compound was tested in parallel applications that highlight the metal-chelating nature of the compound. In the first application, Ybt was assessed as a rust remover, demonstrating a capacity of ∼40% compared to a commercial removal agent and ∼20% relative to total removal capacity. The second application tested Ybt in removing copper from a variety of nonbiological and biological solution mixtures. Success across a variety of media indicates potential utility in diverse scenarios that include environmental and biomedical settings. PMID:26025901

Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin.

PubMed

Zhou, Yongjun; Murphy, Annabel C; Samborskyy, Markiyan; Prediger, Patricia; Dias, Luiz Carlos; Leadlay, Peter F

2015-06-18

Conglobatin is an unusual C2-symmetrical macrodiolide from the bacterium Streptomyces conglobatus with promising antitumor activity. Insights into the genes and enzymes that govern both the assembly-line production of the conglobatin polyketide and its dimerization are essential to allow rational alterations to be made to the conglobatin structure. We have used a rapid, direct in vitro cloning method to obtain the entire cluster on a 41-kbp fragment, encoding a modular polyketide synthase assembly line. The cloned cluster directs conglobatin biosynthesis in a heterologous host strain. Using a model substrate to mimic the conglobatin monomer, we also show that the conglobatin cyclase/thioesterase acts iteratively, ligating two monomers head-to-tail then re-binding the dimer product and cyclizing it. Incubation of two different monomers with the cyclase produces hybrid dimers and trimers, providing the first evidence that conglobatin analogs may in future become accessible through engineering of the polyketide synthase. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Inhibition of Aspergillus parasiticus and cancer cells by marine actinomycete strains

NASA Astrophysics Data System (ADS)

Li, Ping; Yan, Peisheng

2014-12-01

Ten actinomycete strains isolated from the Yellow Sea off China's coasts were identified as belonging to two genera by 16S rDNA phylogenetic analysis: Streptomyces and Nocardiopsis. Six Streptomyces strains (MA10, 2SHXF01-3, MA35, MA05-2, MA05-2-1 and MA08-1) and one Nocardiopsis strain (MA03) were predicted to have the potential to produce aromatic polyketides based on the analysis of the KSα (ketoacyl-synthase) gene in the type II PKS (polyketides synthase) gene cluster. Four strains (MA03, MA01, MA10 and MA05-2) exhibited significant inhibitory effects on mycelia growth (inhibition rate >50%) and subsequent aflatoxin production (inhibition rate >75%) of the mutant aflatoxigenic Aspergillus parasiticus NFRI-95. The ethyl acetate extracts of the broth of these four strains displayed significant inhibitory effects on mycelia growth, and the IC50 values were calculated (MA03: 0.275 mg mL-1, MA01: 0.106 mg mL-1, MA10: 1.345 mg mL-1 and MA05-2: 1.362 mg mL-1). Five strains (2SHXF01-3, MA03, MA05-2, MA01 and MA08-1) were selected based on their high cytotoxic activities. The ethyl acetate extract of the Nocardiopsis strain MA03 was particularly noted for its high antitumor activity against human carcinomas of the cervix (HeLa), lung (A549), kidney (Caki-1) and liver (HepG2) (IC50: 2.890, 1.981, 3.032 and 2.603 μg mL-1, respectively). The extract also remarkably inhibited colony formation of HeLa cells at an extremely low concentration (0.5 μg mL-1). This study highlights that marine-derived actinomycetes are a huge resource of compounds for the biological control of aflatoxin contamination and the development of novel drugs for human carcinomas.

Structure and function of polyketide biosynthetic enzymes: various strategies for production of structurally diverse polyketides.

PubMed

Miyanaga, Akimasa

2017-12-01

Polyketides constitute a large family of natural products that display various biological activities. Polyketides exhibit a high degree of structural diversity, although they are synthesized from simple acyl building blocks. Recent biochemical and structural studies provide a better understanding of the biosynthetic logic of polyketide diversity. This review highlights the biosynthetic mechanisms of structurally unique polyketides, β-amino acid-containing macrolactams, enterocin, and phenolic lipids. Functional and structural studies of macrolactam biosynthetic enzymes have revealed the unique biosynthetic machinery used for selective incorporation of a rare β-amino acid starter unit into the polyketide skeleton. Biochemical and structural studies of cyclization enzymes involved in the biosynthesis of enterocin and phenolic lipids provide mechanistic insights into how these enzymes diversify the carbon skeletons of their products.

MCAT is not required for in vitro polyketide synthesis in a minimal actinorhodin polyketide synthase from Streptomyces coelicolor.

PubMed

Matharu, A L; Cox, R J; Crosby, J; Byrom, K J; Simpson, T J

1998-12-01

It has been proposed that Streptomyces malonyl CoA: holo acyl carrier protein transacylases (MCATs) provide a link between fatty acid and polyketide biosynthesis. Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in reconstituted minimal polyketide synthases (PKSs). In contrast to this, we previously showed that the holo acyl carrier proteins (ACPs) from type II PKSs are capable of catalytic self-malonylation in the presence of malonyl CoA, which suggests that MCAT might not be necessary for polyketide biosynthesis. We reconstituted a homologous actinorhodin (act) type II minimal PKS in vitro. When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed. When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT. The rate of polyketide production increases with holo-ACP concentration, but at low ACP concentration or equimolar AC:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentrations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex. The rate of polyketide biosynthesis is dictated by the ratio of holo-ACP to KS and CLF, as well as by the total protein concentration. There is no absolute requirement for MCAT in polyketide biosynthesis in vitro, although the role of MCAT during polyketide synthesis in vivo remains an open question. MCAT might be responsible for the rate enhancement of malonyl transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP.

Botrydial and botcinins produced by Botrytis cinerea regulate expression of Trichoderma arundinaceum genes involved in trichothecene biosynthesis

USDA-ARS?s Scientific Manuscript database

Trichoderma arundinaceum (Ta37) and Botrytis cinerea produce the sesquiterpenes harzianum A (HA) and botrydial (BOT), respectively, and also the polyketides aspinolides (Asp) and botcinines (Botc), respectively. In the present work, we analyzed the role of BOT and Botcs in the T. arundinaceum-B. cin...

Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

Leveraging microbial biosynthetic pathways for the generation of ‘drop-in’ biofuels

DOE PAGES

Zargar, Amin; Bailey, Constance B.; Haushalter, Robert W.; ...

2017-04-17

Advances in retooling microorganisms have enabled bioproduction of ‘drop-in’ biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gasturbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), ‘drop-in’ biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical propertiesmore » (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel.« less

Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B 2 Biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barajas, Jesus F.; Zargar, Amin; Pang, Bo

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. While β-amino acid loading in the fluvirucin B 2 polyketide pathway has been proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. In this paper, we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B 2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferasesmore » to selectively transfer β-amino acids onto a PKS loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.« less

Leveraging microbial biosynthetic pathways for the generation of 'drop-in' biofuels.

PubMed

Zargar, Amin; Bailey, Constance B; Haushalter, Robert W; Eiben, Christopher B; Katz, Leonard; Keasling, Jay D

2017-06-01

Advances in retooling microorganisms have enabled bioproduction of 'drop-in' biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gas-turbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), 'drop-in' biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical properties (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biochemical Characterization of β-Amino Acid Incorporation in Fluvirucin B 2 Biosynthesis

DOE PAGES

Barajas, Jesus F.; Zargar, Amin; Pang, Bo; ...

2018-03-30

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. While β-amino acid loading in the fluvirucin B 2 polyketide pathway has been proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. In this paper, we elucidate the complete biosynthetic pathway of the β-amino acid loading pathway in fluvirucin B 2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferasesmore » to selectively transfer β-amino acids onto a PKS loading platform. The results presented here provide a detailed biochemical description of β-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.« less

Combined genetic and bioactivity-based prioritization leads to the isolation of an endophyte-derived antimycobacterial compound.

PubMed

Alvin, A; Kalaitzis, J A; Sasia, B; Neilan, B A

2016-05-01

To initiate a genetic and bioactivity-based screening programme of culturable endophytes to identify micro-organisms capable of producing bioactive polyketides and peptides. Fungal endophytes were isolated from flowers, leaves and roots of Rhoeo spathacea, revealing a community consisting of Colletotrichum sp., Fusarium sp., Guignardia sp., Phomopsis sp., Phoma sp. and Microdochium sp. Genetic screening showed that all isolates had polyketide synthase (PKS) genes and most had nonribosomal peptide synthetase (NRPS) genes. Ethyl acetate extracts of the fungal isolates exhibited antiproliferative activity against at least one of the seven bacterial and mycobacterial test strains. Nuclear Magnetic Resonance -guided fractionation of the crude extract from a Fusarium sp. strain which exhibited strong antiproliferative activity against Mycobacterium tuberculosis resulted in the isolation of the polyketide javanicin. This compound was active against Myco. tuberculosis (MIC = 25 μg ml(-1)) and Mycobacterium phlei (MIC = 50 μg ml(-1)). The medicinal plant R. spathacea hosts a variety of fungal endophytes capable of producing antibacterial and antimycobacterial compounds. There is a positive correlation between the presence of PKS and/or NRPS encoding genes in endophytes and the bioactivity of their respective organic extracts. This is the first report on the fungal endophytic diversity of R. spathacea, and the isolation of an antimycobacterial compound from the plant which has been traditionally used for the treatment of tuberculosis symptoms. © 2016 The Society for Applied Microbiology.

Induced systemic resistance (ISR) in Arabidopsis thaliana against Pseudomonas syringae pv. tomato by 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens

USDA-ARS?s Scientific Manuscript database

Pseudomonas fluorescens strains that produce the polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are among the most effective rhizobacteria that suppress root and crown rots, wilts and damping-off diseases of a variety of crops, and they play a key role in the natural suppressiveness of ...

Draft genome sequence of Streptomyces vitaminophilus ATCC 31673, a producer of pyrrolomycin antibiotics, some of which contain a nitro group

DOE PAGES

Mahan, Kristina M.; Klingeman, Dawn Marie; Robert L. Hettich; ...

2016-01-21

Streptomyces vitaminophilus produces pyrrolomycins, which are halogenated polyketide antibiotics. Some of the pyrrolomycins contain a rare nitro group located on the pyrrole ring. In addition, the 6.5-Mbp genome encodes 5,941 predicted protein-coding sequences in 39 contigs with a 71.9% G+C content.

Draft Genome Sequence of Streptomyces vitaminophilus ATCC 31673, a Producer of Pyrrolomycin Antibiotics, Some of Which Contain a Nitro Group

PubMed Central

Klingeman, Dawn M.; Hettich, Robert L.; Parry, Ronald J.

2016-01-01

Streptomyces vitaminophilus produces pyrrolomycins, which are halogenated polyketide antibiotics. Some of the pyrrolomycins contain a rare nitro group located on the pyrrole ring. The 6.5-Mbp genome encodes 5,941 predicted protein-coding sequences in 39 contigs with a 71.9% G+C content. PMID:26798098

Analysis of transporter responsible for the secretion of fusaric acid from the plant pathogen Fusarium oxysporum f. sp. vasinfectum

USDA-ARS?s Scientific Manuscript database

Fusaric acid (FA), a phytotoxic polyketide produced by Fusarium oxysporum f. sp. vasinfectum (Fov), has been suggested to be associated with disease symptoms on cotton. In response to a potential threat on cotton production by the introduction of high FA producing strains from Australia, new sources...

Draft genome sequence of Streptomyces vitaminophilus ATCC 31673, a producer of pyrrolomycin antibiotics, some of which contain a nitro group

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahan, Kristina M.; Klingeman, Dawn Marie; Robert L. Hettich

Streptomyces vitaminophilus produces pyrrolomycins, which are halogenated polyketide antibiotics. Some of the pyrrolomycins contain a rare nitro group located on the pyrrole ring. In addition, the 6.5-Mbp genome encodes 5,941 predicted protein-coding sequences in 39 contigs with a 71.9% G+C content.

A Polyketide Synthase Encoded by the Gene An15g07920 Is Involved in the Biosynthesis of Ochratoxin A in Aspergillus niger.

PubMed

Zhang, Jian; Zhu, Liuyang; Chen, Haoyu; Li, Min; Zhu, Xiaojuan; Gao, Qiang; Wang, Depei; Zhang, Ying

2016-12-28

The polyketide synthase gene An15g07920 was known in Aspergillus niger CBS 513.88 as putatively involved in the production of ochratoxin A (OTA). Genome resequencing analysis revealed that the gene An15g07920 is also present in the ochratoxin-producing A. niger strain 1062. Disruption of An15g07920 in A. niger 1062 removed its capacity to biosynthesize ochratoxin β (OTβ), ochratoxin α (OTα), and OTA. These results indicate that the polyketide synthase encoded by An15g07920 is a crucial player in the biosynthesis of OTA, in the pathway prior to the phenylalanine ligation step. The gene An15g07920 reached its maximum transcription level before OTA accumulation reached its highest level, confirming that gene transcription precedes OTA production. These findings will not only help explain the mechanism of OTA production in A. niger but also provide necessary information for the development of effective diagnostic, preventive, and control strategies to reduce the risk of OTA contamination in foods.

Draft Genome Sequence of Streptomyces vitaminophilus ATCC 31673, a Producer of Pyrrolomycin Antibiotics, Some of Which Contain a Nitro Group.

PubMed

Mahan, Kristina M; Klingeman, Dawn M; Hettich, Robert L; Parry, Ronald J; Graham, David E

2016-01-21

Streptomyces vitaminophilus produces pyrrolomycins, which are halogenated polyketide antibiotics. Some of the pyrrolomycins contain a rare nitro group located on the pyrrole ring. The 6.5-Mbp genome encodes 5,941 predicted protein-coding sequences in 39 contigs with a 71.9% G+C content. Copyright © 2016 Mahan et al.

Draft Genome Sequence of the Rifamycin Producer Amycolatopsis rifamycinica DSM 46095

PubMed Central

Saxena, Anjali; Kumari, Rashmi; Mukherjee, Udita; Singh, Priya

2014-01-01

Amycolatopsis rifamycinica DSM 46095 is an actinobacterium that produces rifamycin SV, an antibiotic used against Mycobacterium tuberculosis. Here, we present the draft genome of DSM 46095, which harbors a novel rifamycin polyketide biosynthetic gene cluster (rif PKS) that differed by 10% in nucleotide sequence from the already reported rif PKS cluster of Amycolatopsis mediterranei S699. PMID:24994803

Quantum chemical study of the structure and properties of citrinin

USDA-ARS?s Scientific Manuscript database

Citrinin is a well known polyketide mycotoxin produced by fungal species that naturally occur in certain agricultural commodities, including red yeast rice. This toxin possesses complex intramolecular hydrogen bonding interactions which influence its mode of action and selective detection. Detailed ...

Improvement of FK506 Production in Streptomyces tsukubaensis by Genetic Enhancement of the Supply of Unusual Polyketide Extender Units via Utilization of Two Distinct Site-Specific Recombination Systems

PubMed Central

Chen, Dandan; Zhang, Qi; Zhang, Qinglin; Cen, Peilin

2012-01-01

FK506 is a potent immunosuppressant that has a wide range of clinical applications. Its 23-member macrocyclic scaffold, mainly with a polyketide origin, features two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, due to the region-specific incorporation of two unusual extender units derived from methoxymalonyl-acyl carrier protein (ACP) and allylmalonyl-coenzyme A (CoA), respectively. Whether their intracellular formations can be a bottleneck for FK506 production remains elusive. In this study, we report the improvement of FK506 yield in the producing strain Streptomyces tsukubaensis by the duplication of two sets of pathway-specific genes individually encoding the biosyntheses of these two extender units, thereby providing a promising approach to generate high-FK506-producing strains via genetic manipulation. Taking advantage of the fact that S. tsukubaensis is amenable to two actinophage (ΦC31 and VWB) integrase-mediated recombination systems, we genetically enhanced the biosyntheses of methoxymalonyl-ACP and allylmalonyl-CoA, as indicated by transcriptional analysis. Together with the optimization of glucose supplementation, the maximal FK506 titer eventually increased by approximately 150% in comparison with that of the original strain. The strategy of engineering the biosynthesis of unusual extender units described here may be applicable to improving the production of other polyketide or nonribosomal peptide natural products that contain pathway-specific building blocks. PMID:22582065

Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis in Streptomyces griseus DSM40695

PubMed Central

Smith, Wyatt C.; Xiang, Longkuan; Shen, Ben

2000-01-01

The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR and nonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of the nonS mutant by the expression of nonS in trans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the nonS mutant in the presence of exogenous (±)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonS gene and fermentation of the nonS mutant in the presence of exogenously added (±)-NA suggests that NonS catalyzes the formation of (−)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in S. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis. PMID:10858335

Natural product diversity associated with the nematode symbionts Photorhabdus and Xenorhabdus

USDA-ARS?s Scientific Manuscript database

Xenorhabdus and Photorhabdus species produce many specialized metabolites derived from non-ribosomal synthetase (NRPS) or polyketide synthase (PKS) with utilities in maintaining a complex life cycle. Both bacteria undergo a symbiosis with nematodes which is then followed by an insect pathogenic phas...

Draft genome sequence of marine-derived Streptomyces sp. TP-A0598, a producer of anti-MRSA antibiotic lydicamycins.

PubMed

Komaki, Hisayuki; Ichikawa, Natsuko; Hosoyama, Akira; Fujita, Nobuyuki; Igarashi, Yasuhiro

2015-01-01

Streptomyces sp. TP-A0598, isolated from seawater, produces lydicamycin, structurally unique type I polyketide bearing two nitrogen-containing five-membered rings, and four congeners TPU-0037-A, -B, -C, and -D. We herein report the 8 Mb draft genome sequence of this strain, together with classification and features of the organism and generation, annotation and analysis of the genome sequence. The genome encodes 7,240 putative ORFs, of which 4,450 ORFs were assigned with COG categories. Also, 66 tRNA genes and one rRNA operon were identified. The genome contains eight gene clusters involved in the production of polyketides and nonribosomal peptides. Among them, a PKS/NRPS gene cluster was assigned to be responsible for lydicamycin biosynthesis and a plausible biosynthetic pathway was proposed on the basis of gene function prediction. This genome sequence data will facilitate to probe the potential of secondary metabolism in marine-derived Streptomyces.

Chemical Probes for the Functionalization of Polyketide Intermediates**

PubMed Central

Riva, Elena; Wilkening, Ina; Gazzola, Silvia; Li, W M Ariel; Smith, Luke; Leadlay, Peter F; Tosin, Manuela

2014-01-01

A library of functionalized chemical probes capable of reacting with ketosynthase-bound biosynthetic intermediates was prepared and utilized to explore in vivo polyketide diversification. Fermentation of ACP mutants of S. lasaliensis in the presence of the probes generated a range of unnatural polyketide derivatives, including novel putative lasalocid A derivatives characterized by variable aryl ketone moieties and linear polyketide chains (bearing alkyne/azide handles and fluorine) flanking the polyether scaffold. By providing direct information on microorganism tolerance and enzyme processing of unnatural malonyl-ACP analogues, as well as on the amenability of unnatural polyketides to further structural modifications, the chemical probes constitute invaluable tools for the development of novel mutasynthesis and synthetic biology. PMID:25212788

Antimicrobial metabolites from the plant endophytic fungus Penicillium sp.

PubMed

Yang, Ming-Hua; Li, Tian-Xiao; Wang, Ying; Liu, Rui-Huan; Luo, Jun; Kong, Ling-Yi

2017-01-01

Five rare dichloro aromatic polyketides (1-5) were obtained from an endophytic fungus Penicillium sp., along with five known metabolites (6-10). Their structures were elucidated by extensive spectroscopic analysis, Mosher methods, as well as [Rh 2 (OCOCF 3 ) 4 ]-induced electronic circular dichroism (ECD) experiments. Compounds 2-4 and 6 structurally involved acyclic 1.3-diols, the uneasy configuration determinations of which were well carried out by double-derivation NMR methods. Compounds 1-10 were evaluated for their antibacterial and antifungal activities against five strains of human pathogenic microorganisms. Helvolic acid (7) showed potent inhibitory effects against Staphylococcus aureus and Pseudomonas aeruginosa with MIC (minimum inhibitory concentration) values of 5.8 and 4.6μg/mL, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line.

PubMed

Ishikawa, Fumihiro; Sugimoto, Hiroyasu; Kakeya, Hideaki

2016-11-17

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl-CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl-CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self-malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self-acylation. This lack of self-malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein-protein interaction between the fatty acid and polyketide synthases in the Adm assembly line. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biochemical and Structural Basis for Controlling Chemical Modularity in Fungal Polyketide Biosynthesis

DOE PAGES

Winter, Jaclyn M.; Cascio, Duilio; Dietrich, David; ...

2015-07-14

Modular collaboration between iterative fungal polyketide synthases (IPKSs) is an important mechanism for generating structural diversity of polyketide natural products. Inter-PKS communication and substrate channeling are controlled in large by the starter unit acyl carrier protein transacylase (SAT) domain found in the accepting IPKS module. Here in this study, we reconstituted the modular biosynthesis of the benzaldehyde core of the chaetoviridin and chaetomugilin azaphilone natural products using the IPKSs CazF and CazM. Our studies revealed a critical role of CazM’s SAT domain in selectively transferring a highly reduced triketide product from CazF. In contrast, a more oxidized triketide that ismore » also produced by CazF and required in later stages of biosynthesis of the final product is not recognized by the SAT domain. The structural basis for the acyl unit selectivity was uncovered by the first X-ray structure of a fungal SAT domain, highlighted by a covalent hexanoyl thioester intermediate in the SAT active site. Finally, the crystal structure of SAT domain will enable protein engineering efforts aimed at mixing and matching different IPKS modules for the biosynthesis of new compounds.« less

The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex.

PubMed

Gay, Darren C; Wagner, Drew T; Meinke, Jessica L; Zogzas, Charles E; Gay, Glen R; Keatinge-Clay, Adrian T

2016-03-01

Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line. Copyright © 2015 Elsevier Inc. All rights reserved.

The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex

PubMed Central

Gay, Darren C.; Wagner, Drew T.; Meinke, Jessica L.; Zogzas, Charles E.; Gay, Glen R.; Keatinge-Clay, Adrian T.

2016-01-01

Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line. PMID:26724270

Evidence of aneuploidy modulating aflatoxigenicity in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Aspergillus flavus is a well-known pathogen of many important agricultural commodities and is a major producer of aflatoxins, which are carcinogenic polyketides that pose a serious health risk to humans and animals. Aflatoxin contamination in peanut exports worldwide accounts for as much as $450 mi...

The Fungal Metabolite, Pyrrocidine A, induces Apoptosis in HEPG2 Hepatocytes and PK15 Renal Cells

USDA-ARS?s Scientific Manuscript database

Pyrrocidines are polyketide-amino acid-derived antibiotics produced by Acremonium zeae, a prevalent seed-borne endophyte of corn. Pyrrocidines exhibit potent activity against Gram-positive bacteria, including drug resistant strains, and display significant activity against Candida albicans, as well...

Antagonistic roles of solanapyrone A during sporulation of the chickpea Ascochyta rabiei

USDA-ARS?s Scientific Manuscript database

Fungi are noted for production of a diverse array of secondary metabolites, many of which are of pharmacological importance. However, the biological roles of these molecules during the fungal life cycle in nature remain elusive. Solanapyrones are polyketide-derived secondary metabolites produced by ...

Stalk cell differentiation without polyketides in the cellular slime mold.

PubMed

Sato, Yukie G; Suarez, Teresa; Saito, Tamao

2016-07-01

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells-the major part of the prestalk cells-are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.

Polyketide biosynthesis in dinoflagellates: what makes it different?

PubMed

Van Wagoner, Ryan M; Satake, Masayuki; Wright, Jeffrey L C

2014-09-01

Dinoflagellates produce unique polyketides characterized by their size and complexity. The biosynthesis of a limited number of such metabolites has been reported, with studies largely hampered by the low yield of compounds and the severe scrambling of label in the isotopically-labeled precursors. Nonetheless, of the successful biosynthetic experiments that have been reported, many surprising and unique processes have been discovered. This knowledge has been accessed through a series of biochemical labeling studies, and while limited molecular genetic data has been amassed, it is still in the early stages of development. In an attempt to meet this challenge, this review has compared some of the biosynthetic processes with similar ones identified in other microbes such as bacteria and myxobacteria, with the idea that similar genes and enzymes are employed by dinoflagellates.

Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5

PubMed Central

Onwueme, Kenolisa C.; Ferreras, Julian A.; Buglino, John; Lima, Christopher D.; Quadri, Luis E. N.

2004-01-01

Mycobacterium tuberculosis (Mt) produces complex virulence-enhancing lipids with scaffolds consisting of phthiocerol and phthiodiolone dimycocerosate esters (PDIMs). Sequence analysis suggested that PapA5, a so-called polyketide-associated protein (Pap) encoded in the PDIM synthesis gene cluster, as well as PapA5 homologs found in Mt and other species, are a subfamily of acyltransferases. Studies with recombinant protein confirmed that PapA5 is an acetyltransferase. Deletion analysis in Mt demonstrated that papA5 is required for PDIM synthesis. We propose that PapA5 catalyzes diesterification of phthiocerol and phthiodiolone with mycocerosate. These studies present the functional characterization of a Pap and permit inferences regarding roles of other Paps in the synthesis of complex lipids, including the antibiotic rifamycin. PMID:15070765

RNAi-mediated down-regulation of a melanin polyketide synthase (pks1) gene in the fungus Slafractonia leguminicola

USDA-ARS?s Scientific Manuscript database

The fungus Slafractonia leguminicola, the causal agent of blackpatch disease of legumes produces two mycotoxins slaframine and swainsonine, causing slobbers’ symptoms and locoism of grazing animals, respectively. The genetics of this important fungus is poorly understood. This work aimed to develop ...

LAE1 regulates expression of multiple secondary metabolite gene clusters in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

The filamentous fungus Fusarium verticillioides can cause disease of maize and is capable of producing the polyketide derived mycotoxins called fumonisins. Fumonisin contamination of maize kernels is a food safety concern. Fumonisins have been implicated in human esophageal cancer as well as in cau...

Identification of early fumonisin biosynthetic intermediates by inactivation of the FUM6 gene in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

Fumonisins are polyketide mycotoxins produced by the maize pathogen Fusarium verticillioides and are associated with multiple human and animal diseases. A fumonisin biosynthetic pathway has been proposed, but structures of early pathway intermediates have not been demonstrated. The F. verticillioide...

Evolution of polyketide synthesis in a Dothideomycete forest pathogen

USDA-ARS?s Scientific Manuscript database

Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in or...

The diversity of anti-microbial secondary metabolites produced by fungal endophytes: an interdisciplinary perspective.

PubMed

Mousa, Walaa Kamel; Raizada, Manish N

2013-01-01

Endophytes are microbes that inhabit host plants without causing disease and are reported to be reservoirs of metabolites that combat microbes and other pathogens. Here we review diverse classes of secondary metabolites, focusing on anti-microbial compounds, synthesized by fungal endophytes including terpenoids, alkaloids, phenylpropanoids, aliphatic compounds, polyketides, and peptides from the interdisciplinary perspectives of biochemistry, genetics, fungal biology, host plant biology, human and plant pathology. Several trends were apparent. First, host plants are often investigated for endophytes when there is prior indigenous knowledge concerning human medicinal uses (e.g., Chinese herbs). However, within their native ecosystems, and where investigated, endophytes were shown to produce compounds that target pathogens of the host plant. In a few examples, both fungal endophytes and their hosts were reported to produce the same compounds. Terpenoids and polyketides are the most purified anti-microbial secondary metabolites from endophytes, while flavonoids and lignans are rare. Examples are provided where fungal genes encoding anti-microbial compounds are clustered on chromosomes. As different genera of fungi can produce the same metabolite, genetic clustering may facilitate sharing of anti-microbial secondary metabolites between fungi. We discuss gaps in the literature and how more interdisciplinary research may lead to new opportunities to develop bio-based commercial products to combat global crop and human pathogens.

The Diversity of Anti-Microbial Secondary Metabolites Produced by Fungal Endophytes: An Interdisciplinary Perspective

PubMed Central

Mousa, Walaa Kamel; Raizada, Manish N.

2013-01-01

Endophytes are microbes that inhabit host plants without causing disease and are reported to be reservoirs of metabolites that combat microbes and other pathogens. Here we review diverse classes of secondary metabolites, focusing on anti-microbial compounds, synthesized by fungal endophytes including terpenoids, alkaloids, phenylpropanoids, aliphatic compounds, polyketides, and peptides from the interdisciplinary perspectives of biochemistry, genetics, fungal biology, host plant biology, human and plant pathology. Several trends were apparent. First, host plants are often investigated for endophytes when there is prior indigenous knowledge concerning human medicinal uses (e.g., Chinese herbs). However, within their native ecosystems, and where investigated, endophytes were shown to produce compounds that target pathogens of the host plant. In a few examples, both fungal endophytes and their hosts were reported to produce the same compounds. Terpenoids and polyketides are the most purified anti-microbial secondary metabolites from endophytes, while flavonoids and lignans are rare. Examples are provided where fungal genes encoding anti-microbial compounds are clustered on chromosomes. As different genera of fungi can produce the same metabolite, genetic clustering may facilitate sharing of anti-microbial secondary metabolites between fungi. We discuss gaps in the literature and how more interdisciplinary research may lead to new opportunities to develop bio-based commercial products to combat global crop and human pathogens. PMID:23543048

Clearing the skies over modular polyketide synthases.

PubMed

Sherman, David H; Smith, Janet L

2006-09-19

Modular polyketide synthases (PKSs) are large multifunctional proteins that synthesize complex polyketide metabolites in microbial cells. A series of recent studies confirm the close protein structural relationship between catalytic domains in the type I mammalian fatty acid synthase (FAS) and the basic synthase unit of the modular PKS. They also establish a remarkable similarity in the overall organization of the type I FAS and the PKS module. This information provides important new conclusions about catalytic domain architecture, function, and molecular recognition that are essential for future efforts to engineer useful polyketide metabolites with valuable biological activities.

An Unusual Fatty Acyl:Adenylate Ligase (FAAL)-Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis.

PubMed

Hemmerling, Franziska; Lebe, Karen E; Wunderlich, Johannes; Hahn, Frank

2018-03-08

The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed by a unique polyene cyclisation mechanism, in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from polyene cyclisation and to transfer it to its associated acyl carrier protein (ACP). After oxidative decarboxylation, the intermediate is channelled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation-thiolation didomain with a very unusual substrate selectivity code but has not yet been biochemically studied. On the basis of sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase (FAAL)-acyl carrier protein didomain with unusual substrate specificity. The expected adenylate-forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalised fatty acids and unsaturated and aromatic carboxylic acids. HPLC-MS analysis and competition experiments show that AmbG preferentially acylates its ACP with long-chain hydrophobic acids and tolerates a π system and a branch near the carboxylic acid. AmbG is the first characterised example of a FAAL-ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as partial reconstitution of the ambruticin pathway to elucidate DVC formation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallo, Antonia; Knox, Benjamin P.; Bruno, Kenneth S.

2014-06-02

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 is composed of 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 amore » 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 which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the β-ketosynthase and acyl-transferase domains of the OTA PKSs which had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both the two 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 qRT-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 which reached its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production.« less

Recent Advances in Microbial Production of Aromatic Chemicals and Derivatives.

PubMed

Noda, Shuhei; Kondo, Akihiko

2017-08-01

Along with the development of metabolic engineering and synthetic biology tools, various microbes are being used to produce aromatic chemicals. In microbes, aromatics are mainly produced via a common important precursor, chorismate, in the shikimate pathway. Natural or non-natural aromatics have been produced by engineering metabolic pathways involving chorismate. In the past decade, novel approaches have appeared to produce various aromatics or to increase their productivity, whereas previously, the targets were mainly aromatic amino acids and the strategy was deregulating feedback inhibition. In this review, we summarize recent studies of microbial production of aromatics based on metabolic engineering approaches. In addition, future perspectives and challenges in this research area are discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Polyketide Derivatives from Annona muricata Linn Leaves as Potencial Anticancer Material by Combination Treatment With Doxorubicin on Hela Cell Line

NASA Astrophysics Data System (ADS)

Artanti, A. N.; Astirin, O. P.; Prayito, A.; Widiyaningsih, R. F.; Prihapsara, F.

2017-02-01

One of the compounds found effication as an anticancer agent on cervical cancer is acetogenin, a polyketide compound that is abundant in Annona muricata L. leaves. This study has been done to examine polyketide derivatives was isolated from Annona muricata L. which has potency to induce apoptosis by p53 expression on hela cell line. An approach recently develop to overcome side effect of chemoterapeutic agent is used of combined chemoterapeutic agent, i.e doxorubicin. The determination of cytotoxic combination activity from polyketide derivative and doxorubicin was evaluated using MTT assay to obtain the value of CI (combination index). The expression of p53 profile was evaluated by immunohistochemistry on hela cell line. Data analysis showed that combination of polyketide derivative from Annona muricata L. (38,5 µg/ml) and doxorubicin with all of concentration performed synergistic effect on hela cell line with CI value from 0,33 - 0,65. The analysis on immucytochemistry showed that polyketide derivative from Annona muricata L. leaves could enhance p53 pathway significantly on hela cell line.

Antibacterial polyketides from Bacillus amyloliquefaciens associated with edible red seaweed Laurenciae papillosa.

PubMed

Chakraborty, Kajal; Thilakan, Bini; Raola, Vamshi Krishna; Joy, Minju

2017-03-01

Heterotrophic Bacillus amyloliquefaciens associated with edible red seaweed, Laurenciae papillosa was used to isolate antibacterial polyketide compounds. Antibacterial activity studies integrated with the outcome obtained by polyketide synthetase (pks) coding genes established that seaweed-affiliated bacterial flora had a wide-ranging antibacterial activities and potential natural product diversity, which proved that the bacterium is valuable reservoir of novel bioactive metabolites. Bioactivity-guided isolation of 3-(octahydro-9-isopropyl-2H-benzo[h]chromen-4-yl)-2-methylpropyl benzoate and methyl 8-(2-(benzoyloxy)-ethyl)-hexahydro-4-((E)-pent-2-enyl)-2H-chromene-6-carboxylate of polyketide origin, with activity against human opportunistic food pathogenic microbes, have been isolated from the ethyl acetate extract of B. amyloliquefaciens. Structure-activity relationship analysis revealed that hydrophobic descriptor of the polyketide compounds significantly contribute towards its antibacterial activity. Seaweed-associated microorganisms were shown to represent a potential source of antimicrobial compounds for food and health benefits. The antibacterial polyketide compounds described in the present study may find potential applications in the food industry to reduce food-borne pathogens. Copyright © 2016 Elsevier Ltd. All rights reserved.

Volatiles from the fungal microbiome of the marine sponge Callyspongia cf. flammea.

PubMed

Barra, Lena; Barac, Paul; König, Gabriele M; Crüsemann, Max; Dickschat, Jeroen S

2017-09-13

The volatiles emitted by five fungal strains previously isolated from the marine sponge Callyspongia cf. flammea were captured with a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Besides several widespread compounds, a series of metabolites with interesting bioactivities were found, including the quorum sensing inhibitor protoanemonin, the fungal phytotoxin 3,4-dimethylpentan-4-olide, and the insect attractant 1,2,4-trimethoxybenzene. In addition, the aromatic polyketides isotorquatone and chartabomone that are both known from Eucalyptus and a new O-desmethyl derivative were identified. The biosynthesis of isotorquatone was studied by feeding experiments with isotopically labeled precursors and its absolute configuration was determined by enantioselective synthesis of a reference compound. Bioactivity testings showed algicidal activity for some of the identified compounds, suggesting a potential ecological function in sponge defence.

The type I fatty acid and polyketide synthases: a tale of two megasynthases

PubMed Central

Tsai, Shiou-Chuan

2008-01-01

This review chronicles the synergistic growth of the fields of fatty acid and polyketide synthesis over the last century. In both animal fatty acid synthases and modular polyketide synthases, similar catalytic elements are covalently linked in the same order in megasynthases. Whereas in fatty acid synthases the basic elements of the design remain immutable, guaranteeing the faithful production of saturated fatty acids, in the modular polyketide synthases, the potential of the basic design has been exploited to the full for the elaboration of a wide range of secondary metabolites of extraordinary structural diversity. PMID:17898897

Identification of a hybrid PKS-NRPS required for the biosynthesis of NG-391 in Metarhizium anisopliae var. anisopliae

USDA-ARS?s Scientific Manuscript database

A 19,818 kb genomic region harboring six predicted ORFs was identified in M. anisopliae ARSEF 2575. ORF4, putatively encoding a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) was targeted using Agrobacterium-mediated gene knockout. Homologous recombinants failed to produce det...

Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis.

PubMed

Zhang, Xiaolin; Chen, Zhi; Li, Meng; Wen, Ying; Song, Yuan; Li, Jilun

2006-10-01

Ivermectin, 22, 23-dihydroavermectin B1, is commercially important in human, veterinary medicine, and pesticides. It is currently synthesized by chemical reduction of the double bond between C22 and C23 of avermectins B1, which are a mixture of B1a (>80%) and B1b (<20%) produced by fermentation of Streptomyces avermitilis. The cost of ivermectin is much higher than that of avermectins B1 owing to the necessity of region-specific hydrogenation at C22-C23 of avermectins B1 with rhodium chloride as the catalyst for producing ivermectin. Here we report that ivermectin can be produced directly by fermentation of recombinant strains constructed through targeted genetic engineering of the avermectin polyketide synthase (PKS) in S. avermitilis Olm73-12, which produces only avermectins B and not avermectins A and oligomycin. The DNA region encoding the dehydratase (DH) and ketoreductase (KR) domains of module 2 from the avermectin PKS in S. avermitilis Olm73-12 was replaced by the DNA fragment encoding the DH, enoylreductase, and KR domains from module 4 of the pikromycin PKS of Streptomyces venezuelae ATCC 15439 using a gene replacement vector pXL211. Twenty-seven of mutants were found to produce a small amount of 22, 23-dihydroavermectin B1a and avermectin B1a and B2a by high performance liquid chromatography and liquid chromatography mass spectrometry analysis. This study might provide a route to the low-cost production of ivermectin by fermentation.

The Combined Use of Proteomics and Transcriptomics Reveals a Complex Secondary Metabolite Network in Peperomia obtusifolia.

PubMed

Batista, Andrea N L; Santos-Pinto, José Roberto A Dos; Batista, João M; Souza-Moreira, Tatiana M; Santoni, Mariana M; Zanelli, Cleslei F; Kato, Massuo J; López, Silvia N; Palma, Mario S; Furlan, Maysa

2017-05-26

Peperomia obtusifolia, an ornamental plant from the Piperaceae family, accumulates a series of secondary metabolites with interesting biological properties. From a biosynthesis standpoint, this species produces several benzopyrans derived from orsellinic acid, which is a polyketide typically found in fungi. Additionally, the chiral benzopyrans were reported as racemic and/or as diastereomeric mixtures, which raises questions about the level of enzymatic control in the cyclization step for the formation of the 3,4-dihydro-2H-pyran moiety. Therefore, this article describes the use of shotgun proteomic and transcriptome studies as well as phytochemical profiling for the characterization of the main biosynthesis pathways active in P. obtusifolia. This combined approach resulted in the identification of a series of proteins involved in its secondary metabolism, including tocopherol cyclase and prenyltransferases. The activity of these enzymes was supported by the phytochemical profiling performed in different organs of P. obtusifolia. However, the polyketide synthases possibly involved in the production of orsellinic acid could not be identified, suggesting that orsellinic acid may be produced by endophytes intimately associated with the plant.

Evolution of Chemical Diversity in a Group of Non-Reduced Polyketide Gene Clusters: Using Phylogenetics to Inform the Search for Novel Fungal Natural Products

PubMed Central

Throckmorton, Kurt; Wiemann, Philipp; Keller, Nancy P.

2015-01-01

Fungal polyketides are a diverse class of natural products, or secondary metabolites (SMs), with a wide range of bioactivities often associated with toxicity. Here, we focus on a group of non-reducing polyketide synthases (NR-PKSs) in the fungal phylum Ascomycota that lack a thioesterase domain for product release, group V. Although widespread in ascomycete taxa, this group of NR-PKSs is notably absent in the mycotoxigenic genus Fusarium and, surprisingly, found in genera not known for their secondary metabolite production (e.g., the mycorrhizal genus Oidiodendron, the powdery mildew genus Blumeria, and the causative agent of white-nose syndrome in bats, Pseudogymnoascus destructans). This group of NR-PKSs, in association with the other enzymes encoded by their gene clusters, produces a variety of different chemical classes including naphthacenediones, anthraquinones, benzophenones, grisandienes, and diphenyl ethers. We discuss the modification of and transitions between these chemical classes, the requisite enzymes, and the evolution of the SM gene clusters that encode them. Integrating this information, we predict the likely products of related but uncharacterized SM clusters, and we speculate upon the utility of these classes of SMs as virulence factors or chemical defenses to various plant, animal, and insect pathogens, as well as mutualistic fungi. PMID:26378577

Proteome analysis provides insight into the regulation of bioactive metabolites in Hericium erinaceus.

PubMed

Zeng, Xu; Ling, Hong; Yang, Jianwen; Chen, Juan; Guo, Shunxing

2018-05-05

Hericium erinaceus, a famous edible mushroom, is also a well-known traditional medicinal fungus. To date, a large number of bioactive metabolites with antitumor, antibacterial, and immune-boosting effects were isolated from the free-living mycelium and fruiting body of H. erinaceus. Here we used the proteomic approach to explore proteins involved in the regulation of bioactive metabolites, including terpenoid, polyketide, sterol and etc. RESULTS: Using mass spectrometry, a total of 2543 unique proteins were identified using H. erinaceus genome, of which 2449, 1855, 1533 and 690 proteins were successfully annotated in Nr, KOG, KEGG and GO databases. Among them, 722 proteins were differentially expressed (528 up- and 194 down-regulated) in fruiting body compared with mycelium. Most of differentially expressed proteins were putatively involved in energy metabolism, molecular signaling, and secondary metabolism. Additionally, numerous proteins involved in terpenoid, polyketide, and sterol biosynthesis were identified. Our data revealed that proteins involved in polyketide biosynthesis were up-regulated in the fruiting body, while some proteins in mevalonate (MEP) pathway from terpenoid biosynthesis were generally up-regulated in mycelium. The present study suggested that the differential regulation of biosynthesis genes could produce various bioactive metabolites with pharmacological effects in H. erinaceus. Copyright © 2017. Published by Elsevier B.V.

Probing the Selectivity and Protein•Protein Interactions of a Non-Reducing Fungal Polyketide Synthase Using Mechanism-Based Crosslinkers

PubMed Central

Bruegger, Joel; Haushalter, Bob; Vagstad, Anna; Shakya, Gaurav; Mih, Nathan; Townsend, Craig A.; Burkart, Michael D.; Tsai, Shiou-Chuan

2013-01-01

SUMMARY Protein•protein interactions, which often involve interactions between an acyl carrier protein (ACP) and its partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal non-reducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ a mechanism-based crosslinker to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP•KS interactions, and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediate key interactions with the negatively-charged ACP surface. Such complementary/matching contact pairs can serve as “adapter surfaces” for future efforts to generate new polyketides using NR-PKSs. PMID:23993461

Polyketides, toxins and pigments in Penicillium marneffei.

PubMed

Tam, Emily W T; Tsang, Chi-Ching; Lau, Susanna K P; Woo, Patrick C Y

2015-10-30

Penicillium marneffei (synonym: Talaromyces marneffei) is the most important pathogenic thermally dimorphic fungus in China and Southeastern Asia. The HIV/AIDS pandemic, particularly in China and other Southeast Asian countries, has led to the emergence of P. marneffei infection as an important AIDS-defining condition. Recently, we published the genome sequence of P. marneffei. In the P. marneffei genome, 23 polyketide synthase genes and two polyketide synthase-non-ribosomal peptide synthase hybrid genes were identified. This number is much higher than those of Coccidioides immitis and Histoplasma capsulatum, important pathogenic thermally dimorphic fungi in the Western world. Phylogenetically, these polyketide synthase genes were distributed evenly with their counterparts found in Aspergillus species and other fungi, suggesting that polyketide synthases in P. marneffei did not diverge from lineage-specific gene duplication through a recent expansion. Gene knockdown experiments and ultra-high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry analysis confirmed that at least four of the polyketide synthase genes were involved in the biosynthesis of various pigments in P. marneffei, including melanin, mitorubrinic acid, mitorubrinol, monascorubrin, rubropunctatin, citrinin and ankaflavin, some of which were mycotoxins and virulence factors of the fungus.

Structural and Biochemical Analyses of Regio- and Stereo-Specificities Observed in a Type II Polyketide Ketoreductase

PubMed Central

Javidpour, Pouya; Korman, Tyler Paz; Shakya, Gaurav; Tsai, Shiou-Chuan

2011-01-01

Type II polyketides include antibiotics such as tetracycline, and chemotherapeutics such as daunorubicin. Type II polyketides are biosynthesized by the type II polyketide synthase (PKS) that consists of 5 – 10 stand-alone domains. In many type II PKSs, the type II ketoreductase (KR) specifically reduce the C9-carbonyl group. How the type II KR achieves such a high regio-specificity, and the nature of stereo-specificity, are not well understood. Sequence alignment of KRs led to a hypothesis that a well-conserved 94-XGG-96 motif may be involved in controlling the stereochemistry. The stereo-specificity of single, double and triple mutant combinations of P94L, G95D and G96D were analyzed in vitro and in vivo for the actinorhodin KR (actKR). The P94L mutation is sufficient to change the stereospecificity of actKR. Binary and ternary crystal structures of both wild type and P94L actKR were solved. Together with assay results, docking simulations, and co-crystal structures, a model for stereochemical control is presented herein that elucidates how type II polyketides are introduced into the substrate pocket such that the C9-carbonyl can be reduced with high regio- and stereo-specificities. The molecular features of actKR important for regio- and stereo-specificities can potentially be applied to biosynthesize new polyketides via protein engineering that rationally controls polyketide ketoreduction. PMID:21506596

Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “Streptomyces carzinostaticus”

PubMed Central

Otsuka, Miyuki; Ichinose, Koji; Fujii, Isao; Ebizuka, Yutaka

2004-01-01

Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by “Streptomyces carzinostaticus” var. F-41. The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized. DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules. Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module. Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in Streptomyces coelicolor CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B. Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis. Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7. This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain. Additionally, the protein encoded by ORF3, located upstream of the PKS genes, closely resembles the FADH2-dependent halogenases involved in the formation of halometabolites. The ORF3 protein could be responsible for the halogenation of NCZs, presenting a unique example of a halogenase involved in the biosynthesis of an aliphatic halometabolite. PMID:15328113

Nonribosomal peptides and polyketides of Burkholderia: new compounds potentially implicated in biocontrol and pharmaceuticals.

PubMed

Esmaeel, Qassim; Pupin, Maude; Jacques, Philippe; Leclère, Valérie

2017-05-25

Bacteria belonging to the genus Burkholderia live in various ecological niches and present a significant role in the environments through the excretion of a wide variety of secondary metabolites including modular nonribosomal peptides (NRPs) and polyketides (PKs). These metabolites represent a widely distributed biomedically and biocontrol important class of natural products including antibiotics, siderophores, and anticancers as well as biopesticides that are considered as a novel source that can be used to defend ecological niche from competitors and to promote plant growth. The aim of this review is to present all NRPs produced or potentially produced by strains of Burkholderia, as NRPs represent a major source of active compounds implicated in biocontrol. The review is a compilation of results from a large screening we have performed on 48 complete sequenced genomes available in NCBI to identify NRPS gene clusters, and data found in the literature mainly because some interesting compounds are produced by strains not yet sequenced. In addition to NRPs, hybrids NRPs/PKs are also included. Specific features about biosynthetic gene clusters and structures of the modular enzymes responsible for the synthesis, the biological activities, and the potential uses in agriculture and pharmaceutical of NRPs and hybrids NRPs/PKs will also be discussed.

Engineer Novel Anticancer Bioagents

DTIC Science & Technology

2009-10-01

Nonribosomally by Bacteria Gene depH is depicted as one of the three post- nonribosomal peptide synthetase (NRPS; dark red)/ polyketide synthase (PKS... polyketide synthase -NRPS pathway for FK228 biosynthesis in C. violaceum no. 968 (Cheng et al., 2007). This pathway would lead to the production of an imme...biosynthesis revealing unprecedented architectural complexity for a hybrid polyketide synthase and nonribosomal peptide synthetase. Chem. Biol. 11, 33–45

Disruptions of the genes involved in lysine biosynthesis, iron acquisition, and secondary metabolisms affect virulence and fitness in Metarhizium robertsii

USDA-ARS?s Scientific Manuscript database

To evaluate the total contribution of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways to M. robertsii fitness and virulence, mutants deleted for mrpptA, a gene required for their activation were generated. 'mrpptA strains failed to produce any of the nonribosomal peptid...

Utilization of high performance liquid chromatography coupled to tandem mass spectrometry for characterization of 8-O-methylbostrycoidin production by species of the fungus Fusarium

USDA-ARS?s Scientific Manuscript database

The pigment, 8-O-methylbostrycoidin is a polyketide metabolite produced by multiple species of the fungus Fusarium that infects plant crops, including maize. A technique was developed for the analysis of 8-O-methylbostrycoidin by high performance liquid chromatography coupled to electrospray ionizat...

Transcriptome analysis of a Ustilago maydis ust1 deletion mutant uncovers involvement of laccase and polyketide synthase genes in spore development

USDA-ARS?s Scientific Manuscript database

Ustilago maydis, causal agent of corn smut disease, is a dimorphic fungus alternating between a saprobic budding haploid, and an obligate pathogenic filamentous dikaryon. Maize responds to U. maydis colonization by producing tumorous structures, and only within these does the fungus sporulate, produ...

An overview of rapamycin: from discovery to future perspectives.

PubMed

Yoo, Young Ji; Kim, Hanseong; Park, Sung Ryeol; Yoon, Yeo Joon

2017-05-01

Rapamycin is an immunosuppressive metabolite produced from several actinomycete species. Besides its immunosuppressive activity, rapamycin and its analogs have additional therapeutic potentials, including antifungal, antitumor, neuroprotective/neuroregenerative, and lifespan extension activities. The core structure of rapamycin is derived from (4R,5R)-4,5-dihydrocyclohex-1-ene-carboxylic acid that is extended by polyketide synthase. The resulting linear polyketide chain is cyclized by incorporating pipecolate and further decorated by post-PKS modification enzymes. Herein, we review the discovery and biological activities of rapamycin as well as its mechanism of action, mechanistic target, biosynthesis, and regulation. In addition, we introduce the many efforts directed at enhancing the production of rapamycin and generating diverse analogs and also explore future perspectives in rapamycin research. This review will also emphasize the remarkable pilot studies on the biosynthesis and production improvement of rapamycin by Dr. Demain, one of the world's distinguished scientists in industrial microbiology and biotechnology.

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine.

PubMed

Eustáquio, Alessandra S; McGlinchey, Ryan P; Liu, Yuan; Hazzard, Christopher; Beer, Laura L; Florova, Galina; Alhamadsheh, Mamoun M; Lechner, Anna; Kale, Andrew J; Kobayashi, Yoshihisa; Reynolds, Kevin A; Moore, Bradley S

2009-07-28

Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5'-chloro-5'-deoxyadenosine (5'-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5'-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides.

PubMed

Asai, Teigo; Tsukada, Kento; Ise, Satomi; Shirata, Naoki; Hashimoto, Makoto; Fujii, Isao; Gomi, Katsuya; Nakagawara, Kosuke; Kodama, Eiichi N; Oshima, Yoshiteru

2015-09-01

The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides

NASA Astrophysics Data System (ADS)

Asai, Teigo; Tsukada, Kento; Ise, Satomi; Shirata, Naoki; Hashimoto, Makoto; Fujii, Isao; Gomi, Katsuya; Nakagawara, Kosuke; Kodama, Eiichi N.; Oshima, Yoshiteru

2015-09-01

The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

The Design of a Molecular Assembly Line Based on Biological Molecules

DTIC Science & Technology

2003-06-01

and will demonstrate how one can construct a purely synthetic analogue of a polyketide synthase . 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF...scaffold in programmed assembly and molecular electronics. It is based on the principles of the biological molecules polyketide synthase and kinesin, and in...stereoselective centers) with any reasonable yield, not including the R&D and process development time. Figure 1.6 shows how a polyketide synthase

Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases.

PubMed

Xu, Yuquan; Zhou, Tong; Zhang, Shuwei; Espinosa-Artiles, Patricia; Wang, Luoyi; Zhang, Wei; Lin, Min; Gunatilaka, A A Leslie; Zhan, Jixun; Molnár, István

2014-08-26

Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.

Implication of PKS type I gene and chromatographic strategy for the biodiscovery of antimicrobial polyketide metabolites from endosymbiotic Nocardiopsis prasina CLA68

NASA Astrophysics Data System (ADS)

Rao, H. C. Yashavantha; Rakshith, Devaraju; Gurudatt, D. M.; Satish, Sreedharamurthy

2016-06-01

Advanced approach in probing for polyketide antimicrobials requires novel genomics and chromatographic strategies. An endophytic strain CLA68 was isolated from the root of Combretum latifolium Blume (Combretaceae) collected from the Western Ghats of Southern India. Strain CLA68 was then identified as Nocardiopsis prasina by its characteristic culture morphology and analysis of 16S rRNA gene sequence. Biosynthetic polyketide synthase genes were investigated using two pairs of degenerate primers. Ethyl acetate extract of CLA68 exhibited broad spectrum activity against a panel of test human pathogens. PKS type-I gene detection and chromatographic strategy yielded a robust polyketide antimicrobial compound which identified as nocapyrone E. Minimum inhibitory concentration of the purified compound against MRSA and other human pathogens ranged between 25 and 100 μg/ml. The present work highlights the utility of N. prasina CLA68 as potential source for antimicrobial polyketide nocapyrone E which could help to combat multidrug-resistant pathogens. This study demonstrates feasibility of PKS type-I gene-based molecular approach and chemical investigation by chromatographic approach is the best method for prediction and rapid discovery of novel polyketides from endosymbiotic actinomycetes. The sequence data of this endosymbiotic actinomycete is deposited in GenBank under the accession no. KP269077.

Potential Antiviral Agents from Marine Fungi: An Overview

PubMed Central

Zorofchian Moghadamtousi, Soheil; Nikzad, Sonia; Abdul Kadir, Habsah; Abubakar, Sazaly; Zandi, Keivan

2015-01-01

Biodiversity of the marine world is only partially subjected to detailed scientific scrutiny in comparison to terrestrial life. Life in the marine world depends heavily on marine fungi scavenging the oceans of lifeless plants and animals and entering them into the nutrient cycle by. Approximately 150 to 200 new compounds, including alkaloids, sesquiterpenes, polyketides, and aromatic compounds, are identified from marine fungi annually. In recent years, numerous investigations demonstrated the tremendous potential of marine fungi as a promising source to develop new antivirals against different important viruses, including herpes simplex viruses, the human immunodeficiency virus, and the influenza virus. Various genera of marine fungi such as Aspergillus, Penicillium, Cladosporium, and Fusarium were subjected to compound isolation and antiviral studies, which led to an illustration of the strong antiviral activity of a variety of marine fungi-derived compounds. The present review strives to summarize all available knowledge on active compounds isolated from marine fungi with antiviral activity. PMID:26204947

Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuzawa, Satoshi; Deng, Kai; Wang, George

2016-08-22

Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed in this paper the segments of AT domains and associated linkers in AT exchanges in vitro andmore » have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. Finally, these results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production.« less

Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills.

PubMed

Elshahawi, Sherif I; Trindade-Silva, Amaro E; Hanora, Amro; Han, Andrew W; Flores, Malem S; Vizzoni, Vinicius; Schrago, Carlos G; Soares, Carlos A; Concepcion, Gisela P; Distel, Dan L; Schmidt, Eric W; Haygood, Margo G

2013-01-22

Shipworms are marine wood-boring bivalve mollusks (family Teredinidae) that harbor a community of closely related Gammaproteobacteria as intracellular endosymbionts in their gills. These symbionts have been proposed to assist the shipworm host in cellulose digestion and have been shown to play a role in nitrogen fixation. The genome of one strain of Teredinibacter turnerae, the first shipworm symbiont to be cultivated, was sequenced, revealing potential as a rich source of polyketides and nonribosomal peptides. Bioassay-guided fractionation led to the isolation and identification of two macrodioloide polyketides belonging to the tartrolon class. Both compounds were found to possess antibacterial properties, and the major compound was found to inhibit other shipworm symbiont strains and various pathogenic bacteria. The gene cluster responsible for the synthesis of these compounds was identified and characterized, and the ketosynthase domains were analyzed phylogenetically. Reverse-transcription PCR in addition to liquid chromatography and high-resolution mass spectrometry and tandem mass spectrometry revealed the transcription of these genes and the presence of the compounds in the shipworm, suggesting that the gene cluster is expressed in vivo and that the compounds may fulfill a specific function for the shipworm host. This study reports tartrolon polyketides from a shipworm symbiont and unveils the biosynthetic gene cluster of a member of this class of compounds, which might reveal the mechanism by which these bioactive metabolites are biosynthesized.

Composition of dissolved organic matter in groundwater

NASA Astrophysics Data System (ADS)

Longnecker, Krista; Kujawinski, Elizabeth B.

2011-05-01

Groundwater constitutes a globally important source of freshwater for drinking water and other agricultural and industrial purposes, and is a prominent source of freshwater flowing into the coastal ocean. Therefore, understanding the chemical components of groundwater is relevant to both coastal and inland communities. We used electrospray ionization coupled with Fourier-transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to examine dissolved organic compounds in groundwater prior to and after passage through a sediment-filled column containing microorganisms. The data revealed that an unexpectedly high proportion of organic compounds contained nitrogen and sulfur, possibly due to transport of surface waters from septic systems and rain events. We matched 292 chemical features, based on measured mass:charge ( m/z) values, to compounds stored in the Kyoto Encyclopedia of Genes and Genomes (KEGG). A subset of these compounds (88) had only one structural isomer in KEGG, thus supporting tentative identification. Most identified elemental formulas were linked with metabolic pathways that produce polyketides or with secondary metabolites produced by plants. The presence of polyketides in groundwater is notable because of their anti-bacterial and anti-cancer properties. However, their relative abundance must be quantified with appropriate analyses to assess any implications for public health.

Method of upgrading oils containing hydroxyaromatic hydrocarbon compounds to highly aromatic gasoline

DOEpatents

Baker, Eddie G.; Elliott, Douglas C.

1993-01-01

The present invention is a multi-stepped method of converting an oil which is produced by various biomass and coal conversion processes and contains primarily single and multiple ring hydroxyaromatic hydrocarbon compounds to highly aromatic gasoline. The single and multiple ring hydroxyaromatic hydrocarbon compounds in a raw oil material are first deoxygenated to produce a deoxygenated oil material containing single and multiple ring aromatic compounds. Then, water is removed from the deoxygenated oil material. The next step is distillation to remove the single ring aromatic compouns as gasoline. In the third step, the multiple ring aromatics remaining in the deoxygenated oil material are cracked in the presence of hydrogen to produce a cracked oil material containing single ring aromatic compounds. Finally, the cracked oil material is then distilled to remove the single ring aromatics as gasoline.

Method of upgrading oils containing hydroxyaromatic hydrocarbon compounds to highly aromatic gasoline

DOEpatents

Baker, E.G.; Elliott, D.C.

1993-01-19

The present invention is a multi-stepped method of converting an oil which is produced by various biomass and coal conversion processes and contains primarily single and multiple ring hydroxyaromatic hydrocarbon compounds to highly aromatic gasoline. The single and multiple ring hydroxyaromatic hydrocarbon compounds in a raw oil material are first deoxygenated to produce a deoxygenated oil material containing single and multiple ring aromatic compounds. Then, water is removed from the deoxygenated oil material. The next step is distillation to remove the single ring aromatic compounds as gasoline. In the third step, the multiple ring aromatics remaining in the deoxygenated oil material are cracked in the presence of hydrogen to produce a cracked oil material containing single ring aromatic compounds. Finally, the cracked oil material is then distilled to remove the single ring aromatics as gasoline.

Antibacterial polyketides from the jellyfish-derived fungus Paecilomyces variotii.

PubMed

Liu, Juan; Li, Famei; Kim, Eun La; Li, Jian Lin; Hong, Jongki; Bae, Kyung Sook; Chung, Hae Young; Kim, Hyung Sik; Jung, Jee H

2011-08-26

Four new polyketides (1-4) were isolated from the fungus Paecilomyces variotii, which was derived from the jellyfish Nemopilema nomurai. The planar structures and relative configurations of these polyketides were elucidated on the basis of spectroscopic analyses, including 2D NMR experiments. The compounds showed inhibitory activity against pathogenic bacteria including methicillin-resistant Staphylococcus aureus 3089 and multi-drug-resistant Vibrio parahemolyticus 7001 with MIC values in the range 5-40 μg/mL.

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis.

PubMed

Cardenas, Javier; Da Silva, Nancy A

2016-07-01

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP(+) for acetyl-CoA production. After 24h of cultivation, a 3.7-fold increase in NADPH/NADP(+) ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2-3-fold over the base strain (up to 0.8g/L), and in combination to 1.4g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Production and New Extraction Method of Polyketide Red Pigments Produced by Ascomycetous Fungi from Terrestrial and Marine Habitats

PubMed Central

Lebeau, Juliana; Venkatachalam, Mekala; Fouillaud, Mireille; Petit, Thomas; Vinale, Francesco; Dufossé, Laurent; Caro, Yanis

2017-01-01

The use of ascomycetous fungi as pigment producers opens the way to an alternative to synthetic dyes, especially in the red-dye industries, which have very few natural pigment alternatives. The present paper aimed to bio-prospect and screen out 15 selected ascomycetous fungal strains, originating from terrestrial and marine habitats belonging to seven different genera (Penicillium, Talaromyces, Fusarium, Aspergillus, Trichoderma, Dreschlera, and Paecilomyces). We identified four strains, Penicillium purpurogenum rubisclerotium, Fusarium oxysporum, marine strains identified as Talaromyces spp., and Trichoderma atroviride, as potential red pigment producers. The extraction of the pigments is a crucial step, whereby the qualitative and quantitative compositions of each fungal extract need to be respected for reliable identification, as well as preserving bioactivity. Furthermore, there is a growing demand for more sustainable and cost-effective extraction methods. Therefore, a pressurized liquid extraction technique was carried out in this study, allowing a greener and faster extraction step of the pigments, while preserving their chemical structures and bioactivities in comparison to conventional extraction processes. The protocol was illustrated with the production of pigment extracts from P. purpurogenum rubisclerotium and Talaromyces spp. Extracts were analyzed by high-performance liquid-chromatography combined with photodiode array-detection (HPLC-DAD) and high-resolution mass spectrometry (UHPLC-HRMS). The more promising strain was the isolate Talaromyces spp. of marine origin. The main polyketide pigment produced by this strain has been characterized as N-threoninerubropunctamine, a non-toxic red Monascus-like azaphilone pigment. PMID:29371552

Synergistic Cytotoxicity Effect by Combination Treatment of Polyketide Derivatives from Annona muricata Linn Leaves and Doxorubicin as Potential Anticancer Material on Raji Cell Line

NASA Astrophysics Data System (ADS)

Artanti, A. N.; Astirin, O. P.; Prayito, A.; Fisma, R.; Prihapsara, F.

2018-03-01

Nasopharynx cancer is one of the most deadly cancer. The main priority of nasopharynx cancer treatment is the use of chemotherapeutic agents, especially doxorubicin. However, doxorubicin might also lead to diverse side effect. An approach recently develop to overcome side effect of doxorubicin is to used of combined chemotherapeutic agent. One of the compounds found effication as an anticancer agent on nasopharynx cancer is acetogenin, a polyketide compound that is abundant in Annona muricata L. leaves. This study has been done to examine polyketide derivatives was isolated from Annona muricata L. which has potency to induce apoptosis by p53 expression on raji cell line. The determination of cytotoxic combination activity from polyketide derivative and doxorubicin was evaluated using MTT assay to obtain the value of CI (combination index). Data analysis showed that combination of polyketide derivative from Annona muricata L. (14,4 µg/ml) and doxorubicin with all of concentration performed synergistic effect on raji cell line with CI value from 0.13 – 0.65.

Alternative Sigma Factor Over-Expression Enables Heterologous Expression of a Type II Polyketide Biosynthetic Pathway in Escherichia coli

PubMed Central

Stevens, David Cole; Conway, Kyle R.; Pearce, Nelson; Villegas-Peñaranda, Luis Roberto; Garza, Anthony G.; Boddy, Christopher N.

2013-01-01

Background Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. Methodology We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. Conclusions We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products. PMID:23724102

A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

NASA Astrophysics Data System (ADS)

Ray, Lauren; Valentic, Timothy R.; Miyazawa, Takeshi; Withall, David M.; Song, Lijiang; Milligan, Jacob C.; Osada, Hiroyuki; Takahashi, Shunji; Tsai, Shiou-Chuan; Challis, Gregory L.

2016-12-01

Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

The enzymology of polyether biosynthesis.

PubMed

Liu, Tiangang; Cane, David E; Deng, Zixin

2009-01-01

Polyether ionophore antibiotics are a special class of polyketides widely used in veterinary medicine, and as food additives in animal husbandry. In this article, we review current knowledge about the mechanism of polyether biosynthesis, and the genetic and biochemical strategies used for its study. Several clear differences distinguish it from traditional type I modular polyketide biosynthesis: polyether backbones are assembled by modular polyketide synthases but are modified by two key enzymes, epoxidase and epoxide hydrolase, to generate the product. All double bonds involved in the oxidative cyclization in the polyketide backbone are of E geometry. Chain release in the polyether biosynthetic pathway requires a special type II thioesterase which specifically hydrolyzes the polyether thioester. All these discoveries should be very helpful for a deep understanding of the biosynthetic mechanism of this class of important natural compounds, and for the targeted engineering of polyether derivatives.

HSQC-TOCSY Fingerprinting-Directed Discovery of Antiplasmodial Polyketides from the Marine Ascidian-Derived Streptomyces sp. (USC-16018).

PubMed

Buedenbender, Larissa; Robertson, Luke P; Lucantoni, Leonardo; Avery, Vicky M; Kurtböke, D İpek; Carroll, Anthony R

2018-05-30

Chemical investigations on the fermentation extract obtained from an ascidian-derived Streptomyces sp. (USC-16018) yielded a new ansamycin polyketide, herbimycin G ( 1 ), as well as a known macrocyclic polyketide, elaiophylin ( 2 ), and four known diketopiperazines ( 3 ⁻ 6 ). The structures of the compounds were elucidated based on 1D/2D NMR and MS data. The absolute configuration of 1 was established by comparison of experimental and predicted electronic circular dichroism (ECD) data. Antiplasmodial activities were tested for the natural products against chloroquine sensitive (3D7) and chloroquine resistant (Dd2) Plasmodium falciparum strains; the two polyketides ( 1 ⁻ 2 ) demonstrated an inhibition of >75% against both parasite strains and while 2 was highly cytotoxic, herbimycin G ( 1 ) showed no cytotoxicity and good predicted water solubility.

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

PubMed

Vickery, Christopher R; Cardenas, Javier; Bowman, Marianne E; Burkart, Michael D; Da Silva, Nancy A; Noel, Joseph P

2018-06-01

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts. © 2018 Wiley Periodicals, Inc.

Putative Monofunctional Type I Polyketide Synthase Units: A Dinoflagellate-Specific Feature?

PubMed Central

Eichholz, Karsten; Beszteri, Bánk; John, Uwe

2012-01-01

Marine dinoflagellates (alveolata) are microalgae of which some cause harmful algal blooms and produce a broad variety of most likely polyketide synthesis derived phycotoxins. Recently, novel polyketide synthesase (PKS) transcripts have been described from the Florida red tide dinoflagellate Karenia brevis (gymnodiniales) which are evolutionarily related to Type I PKS but were apparently expressed as monofunctional proteins, a feature typical of Type II PKS. Here, we investigated expression units of PKS I-like sequences in Alexandrium ostenfeldii (gonyaulacales) and Heterocapsa triquetra (peridiniales) at the transcript and protein level. The five full length transcripts we obtained were all characterized by polyadenylation, a 3′ UTR and the dinoflagellate specific spliced leader sequence at the 5′end. Each of the five transcripts encoded a single ketoacylsynthase (KS) domain showing high similarity to K. brevis KS sequences. The monofunctional structure was also confirmed using dinoflagellate specific KS antibodies in Western Blots. In a maximum likelihood phylogenetic analysis of KS domains from diverse PKSs, dinoflagellate KSs formed a clade placed well within the protist Type I PKS clade between apicomplexa, haptophytes and chlorophytes. These findings indicate that the atypical PKS I structure, i.e., expression as putative monofunctional units, might be a dinoflagellate specific feature. In addition, the sequenced transcripts harbored a previously unknown, apparently dinoflagellate specific conserved N-terminal domain. We discuss the implications of this novel region with regard to the putative monofunctional organization of Type I PKS in dinoflagellates. PMID:23139807

Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa

PubMed Central

Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

2015-01-01

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2–C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure–function relationship and catalytic mechanism of OAC. PMID:26625288

Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

PubMed

Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

2015-12-01

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

Biosynthesis and molecular genetics of polyketides in marine dinoflagellates.

PubMed

Kellmann, Ralf; Stüken, Anke; Orr, Russell J S; Svendsen, Helene M; Jakobsen, Kjetill S

2010-03-31

Marine dinoflagellates are the single most important group of algae that produce toxins, which have a global impact on human activities. The toxins are chemically diverse, and include macrolides, cyclic polyethers, spirolides and purine alkaloids. Whereas there is a multitude of studies describing the pharmacology of these toxins, there is limited or no knowledge regarding the biochemistry and molecular genetics involved in their biosynthesis. Recently, however, exciting advances have been made. Expressed sequence tag sequencing studies have revealed important insights into the transcriptomes of dinoflagellates, whereas other studies have implicated polyketide synthase genes in the biosynthesis of cyclic polyether toxins, and the molecular genetic basis for the biosynthesis of paralytic shellfish toxins has been elucidated in cyanobacteria. This review summarises the recent progress that has been made regarding the unusual genomes of dinoflagellates, the biosynthesis and molecular genetics of dinoflagellate toxins. In addition, the evolution of these metabolic pathways will be discussed, and an outlook for future research and possible applications is provided.

Antifouling and antibacterial polyketides from marine gorgonian coral-associated fungus Penicillium sp. SCSGAF 0023.

PubMed

Bao, Jie; Sun, Yu-Lin; Zhang, Xiao-Yong; Han, Zhuang; Gao, Hai-Chun; He, Fei; Qian, Pei-Yuan; Qi, Shu-Hua

2013-04-01

Two new polyketides, 6,8,5'6'-tetrahydroxy-3'-methylflavone (1) and paecilin C (2), together with six known analogs secalonic acid D (3), secalonic acid B (4) penicillixanthone A (5), emodin (6), citreorosein (7) and isorhodoptilometrin (8) were obtained from a broth of gorgonian coral-associated fungus Penicillium sp. SCSGAF 0023. Compounds 1 and 6-8 had significant antifouling activity against Balanus amphitrite larvae settlement with EC50 values of 6.7, 6.1, 17.9 and 13.7 μg ml(-1), respectively, and 3-5 showed medium antibacterial activity against four tested bacterial strains. This was the first report of antibacterial activity of 3-5 against marine bacteria and antifouling activity of 6-8 against marine biofouling organism's larvae. The results indicated that gorgonian coral-associated fungus Penicillium sp. SCSGAF 0023 strain could produce antifouling and antibacterial compounds that might aid the host gorgonian coral in protection against marine pathogen bacteria, biofouling organisms and other intruders.

Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

NASA Astrophysics Data System (ADS)

Itoh, Takayuki; Tokunaga, Kinya; Matsuda, Yudai; Fujii, Isao; Abe, Ikuro; Ebizuka, Yutaka; Kushiro, Tetsuo

2010-10-01

Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

Biosynthesis and Molecular Genetics of Polyketides in Marine Dinoflagellates

PubMed Central

Kellmann, Ralf; Stüken, Anke; Orr, Russell J. S.; Svendsen, Helene M.; Jakobsen, Kjetill S.

2010-01-01

Marine dinoflagellates are the single most important group of algae that produce toxins, which have a global impact on human activities. The toxins are chemically diverse, and include macrolides, cyclic polyethers, spirolides and purine alkaloids. Whereas there is a multitude of studies describing the pharmacology of these toxins, there is limited or no knowledge regarding the biochemistry and molecular genetics involved in their biosynthesis. Recently, however, exciting advances have been made. Expressed sequence tag sequencing studies have revealed important insights into the transcriptomes of dinoflagellates, whereas other studies have implicated polyketide synthase genes in the biosynthesis of cyclic polyether toxins, and the molecular genetic basis for the biosynthesis of paralytic shellfish toxins has been elucidated in cyanobacteria. This review summarises the recent progress that has been made regarding the unusual genomes of dinoflagellates, the biosynthesis and molecular genetics of dinoflagellate toxins. In addition, the evolution of these metabolic pathways will be discussed, and an outlook for future research and possible applications is provided. PMID:20479965

Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism

PubMed Central

Xie, Zhengzhi; Gang, David R.

2009-01-01

Turmeric is an excellent example of a plant that produces large numbers of metabolites from diverse metabolic pathways or networks. It is hypothesized that these metabolic pathways or networks contain biosynthetic modules, which lead to the formation of metabolite modules—groups of metabolites whose production is co-regulated and biosynthetically linked. To test whether such co-regulated metabolite modules do exist in this plant, metabolic profiling analysis was performed on turmeric rhizome samples that were collected from 16 different growth and development treatments, which had significant impacts on the levels of 249 volatile and non-volatile metabolites that were detected. Importantly, one of the many co-regulated metabolite modules that were indeed readily detected in this analysis contained the three major curcuminoids, whereas many other structurally related diarylheptanoids belonged to separate metabolite modules, as did groups of terpenoids. The existence of these co-regulated metabolite modules supported the hypothesis that the 3-methoxyl groups on the aromatic rings of the curcuminoids are formed before the formation of the heptanoid backbone during the biosynthesis of curcumin and also suggested the involvement of multiple polyketide synthases with different substrate selectivities in the formation of the array of diarylheptanoids detected in turmeric. Similar conclusions about terpenoid biosynthesis could also be made. Thus, discovery and analysis of metabolite modules can be a powerful predictive tool in efforts to understand metabolism in plants. PMID:19073964

In Vitro Biosynthesis of Unnatural Enterocin and Wailupemycin Polyketides¥

PubMed Central

Kalaitzis, John A.; Cheng, Qian; Thomas, Paul M.; Kelleher, Neil L.; Moore, Bradley S.

2009-01-01

Nature has evolved finely tuned strategies to synthesize rare and complex natural products such as the enterocin family of polyketides from the marine bacterium Streptomyces maritimus. Herein we report the directed ex vivo multienzyme syntheses of 24 unnatural 5-deoxyenterocin and wailupemycin F and G analogues, 18 of which are new. We have generated molecular diversity by priming the enterocin biosynthesis enzymes with unnatural substrates and have illustrated further the uniqueness of this type II polyketide synthase by way of exploiting its unusual starter unit biosynthesis pathways. PMID:19215142

In vitro biosynthesis of unnatural enterocin and wailupemycin polyketides.

PubMed

Kalaitzis, John A; Cheng, Qian; Thomas, Paul M; Kelleher, Neil L; Moore, Bradley S

2009-03-27

Nature has evolved finely tuned strategies to synthesize rare and complex natural products such as the enterocin family of polyketides from the marine bacterium Streptomyces maritimus. Herein we report the directed ex vivo multienzyme syntheses of 24 unnatural 5-deoxyenterocin and wailupemycin F and G analogues, 18 of which are new. We have generated molecular diversity by priming the enterocin biosynthesis enzymes with unnatural substrates and have illustrated further the uniqueness of this type II polyketide synthase by way of exploiting its unusual starter unit biosynthesis pathways.

Diverse bacterial PKS sequences derived from okadaic acid-producing dinoflagellates.

PubMed

Perez, Roberto; Liu, Li; Lopez, Jose; An, Tianying; Rein, Kathleen S

2008-05-22

Okadaic acid (OA) and the related dinophysistoxins are isolated from dinoflagellates of the genus Prorocentrum and Dinophysis. Bacteria of the Roseobacter group have been associated with okadaic acid producing dinoflagellates and have been previously implicated in OA production. Analysis of 16S rRNA libraries reveals that Roseobacter are the most abundant bacteria associated with OA producing dinoflagellates of the genus Prorocentrum and are not found in association with non-toxic dinoflagellates. While some polyketide synthase (PKS) genes form a highly supported Prorocentrum clade, most appear to be bacterial, but unrelated to Roseobacter or Alpha-Proteobacterial PKSs or those derived from other Alveolates Karenia brevis or Crytosporidium parvum.

Mechanism of Thioesterase-Catalyzed Chain Release in the Biosynthesis of the Polyether Antibiotic Nanchangmycin

PubMed Central

Liu, Tiangang; Lin, Xin; Zhou, Xiufen; Deng, Zixin; Cane, David E.

2008-01-01

Summary The polyketide backbone of the polyether ionophore antibiotic nanchangmycin (1) is assembled by a modular polyketide synthase in Streptomyces nanchangensis NS3226. The ACP-bound polyketide is thought to undergo a cascade of oxidative cyclizations to generate the characteristic polyether. Deletion of the glycosyl transferase gene nanG5 resulted in accumulation of the corresponding nanchangmycin aglycone (6). The discrete thioesterase NanE exhibited a nearly 17-fold preference for hydrolysis of 4, the N-acetylcysteamine (SNAC) thioester of nanchangmycin, over 7, the corresponding SNAC derivative of the aglycone, consistent with NanE-catalyzed hydrolysis of ACP-bound nanchangmycin being the final step in the biosynthetic pathway. Site directed mutagenesis established that Ser96, His261, and Asp120, the proposed components of the NanE catalytic triad, were all essential for thioesterase activity, while Trp97 was shown to influence the preference for polyether over polyketide substrates. PMID:18482697

Diversity and Antimicrobial Activities of Actinobacteria Isolated from Tropical Mangrove Sediments in Malaysia

PubMed Central

Lee, Learn-Han; Zainal, Nurullhudda; Azman, Adzzie-Shazleen; Eng, Shu-Kee; Goh, Bey-Hing; Yin, Wai-Fong; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan

2014-01-01

The aim of this study was to isolate and identify Actinobacteria from Malaysia mangrove forest and screen them for production of antimicrobial secondary metabolites. Eighty-seven isolates were isolated from soil samples collected at 4 different sites. This is the first report to describe the isolation of Streptomyces, Mycobacterium, Leifsonia, Microbacterium, Sinomonas, Nocardia, Terrabacter, Streptacidiphilus, Micromonospora, Gordonia, and Nocardioides from mangrove in east coast of Malaysia. Of 87 isolates, at least 5 isolates are considered as putative novel taxa. Nine Streptomyces sp. isolates were producing potent antimicrobial secondary metabolites, indicating that Streptomyces isolates are providing high quality metabolites for drug discovery purposes. The discovery of a novel species, Streptomyces pluripotens sp. nov. MUSC 135T that produced potent secondary metabolites inhibiting the growth of MRSA, had provided promising metabolites for drug discovery research. The biosynthetic potential of 87 isolates was investigated by the detection of polyketide synthetase (PKS) and nonribosomal polyketide synthetase (NRPS) genes, the hallmarks of secondary metabolites production. Results showed that many isolates were positive for PKS-I (19.5%), PKS-II (42.5%), and NRPS (5.7%) genes, indicating that mangrove Actinobacteria have significant biosynthetic potential. Our results highlighted that mangrove environment represented a rich reservoir for isolation of Actinobacteria, which are potential sources for discovery of antimicrobial secondary metabolites. PMID:25162061

Diversity and antimicrobial activities of actinobacteria isolated from tropical mangrove sediments in Malaysia.

PubMed

Lee, Learn-Han; Zainal, Nurullhudda; Azman, Adzzie-Shazleen; Eng, Shu-Kee; Goh, Bey-Hing; Yin, Wai-Fong; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan

2014-01-01

The aim of this study was to isolate and identify Actinobacteria from Malaysia mangrove forest and screen them for production of antimicrobial secondary metabolites. Eighty-seven isolates were isolated from soil samples collected at 4 different sites. This is the first report to describe the isolation of Streptomyces, Mycobacterium, Leifsonia, Microbacterium, Sinomonas, Nocardia, Terrabacter, Streptacidiphilus, Micromonospora, Gordonia, and Nocardioides from mangrove in east coast of Malaysia. Of 87 isolates, at least 5 isolates are considered as putative novel taxa. Nine Streptomyces sp. isolates were producing potent antimicrobial secondary metabolites, indicating that Streptomyces isolates are providing high quality metabolites for drug discovery purposes. The discovery of a novel species, Streptomyces pluripotens sp. nov. MUSC 135(T) that produced potent secondary metabolites inhibiting the growth of MRSA, had provided promising metabolites for drug discovery research. The biosynthetic potential of 87 isolates was investigated by the detection of polyketide synthetase (PKS) and nonribosomal polyketide synthetase (NRPS) genes, the hallmarks of secondary metabolites production. Results showed that many isolates were positive for PKS-I (19.5%), PKS-II (42.5%), and NRPS (5.7%) genes, indicating that mangrove Actinobacteria have significant biosynthetic potential. Our results highlighted that mangrove environment represented a rich reservoir for isolation of Actinobacteria, which are potential sources for discovery of antimicrobial secondary metabolites.

Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

DOEpatents

Smith, R.E.; Dolbeare, F.A.

1980-10-21

Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes. No Drawings

Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

DOEpatents

Smith, Robert E. [557 Escondido Cir., Livermore, CA 94550; Dolbeare, Frank A. [5178 Diane La., Livermore, CA 94550

1980-10-21

Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 4-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes.

Fluorescence method for enzyme analysis which couples aromatic amines with aromatic aldehydes

DOEpatents

Smith, Robert E.; Dolbeare, Frank A.

1979-01-01

Analysis of proteinases is accomplished using conventional amino acid containing aromatic amine substrates. Aromatic amines such as 4-methoxy-2-naphthylamine (4M2NA), 2-naphthylamine, aminoisophthalic acid dimethyl ester, p-nitroaniline, 5-methoxy-1-aminofluorene and coumarin derivatives resulting from enzymatic hydrolysis of the substrate couples with aromatic aldehydes such as 5-nitrosalicylaldehyde (5-NSA), benzaldehyde and p-nitrobenzaldehyde to produce Schiff-base complexes which are water insoluble. Certain Schiff-base complexes produce a shift from blue to orange-red (visible) fluorescence. Such complexes are useful in the assay of enzymes.

Metabolic engineering of Pseudomonas putida for production of docosahexaenoic acid based on a myxobacterial PUFA synthase.

PubMed

Gemperlein, Katja; Zipf, Gregor; Bernauer, Hubert S; Müller, Rolf; Wenzel, Silke C

2016-01-01

Long-chain polyunsaturated fatty acids (LC-PUFAs) can be produced de novo via polyketide synthase-like enzymes known as PUFA synthases, which are encoded by pfa biosynthetic gene clusters originally discovered from marine microorganisms. Recently similar gene clusters were detected and characterized in terrestrial myxobacteria revealing several striking differences. As the identified myxobacterial producers are difficult to handle genetically and grow very slowly we aimed to establish heterologous expression platforms for myxobacterial PUFA synthases. Here we report the heterologous expression of the pfa gene cluster from Aetherobacter fasciculatus (SBSr002) in the phylogenetically distant model host bacteria Escherichia coli and Pseudomonas putida. The latter host turned out to be the more promising PUFA producer revealing higher production rates of n-6 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). After several rounds of genetic engineering of expression plasmids combined with metabolic engineering of P. putida, DHA production yields were eventually increased more than threefold. Additionally, we applied synthetic biology approaches to redesign and construct artificial versions of the A. fasciculatus pfa gene cluster, which to the best of our knowledge represents the first example of a polyketide-like biosynthetic gene cluster modulated and synthesized for P. putida. Combination with the engineering efforts described above led to a further increase in LC-PUFA production yields. The established production platform based on synthetic DNA now sets the stage for flexible engineering of the complex PUFA synthase. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Catalytic fast co-pyrolysis of biomass and food waste to produce aromatics: Analytical Py-GC/MS study.

PubMed

Zhang, Bo; Zhong, Zhaoping; Min, Min; Ding, Kuan; Xie, Qinglong; Ruan, Roger

2015-01-01

In this study, catalytic fast co-pyrolysis (co-CFP) of corn stalk and food waste (FW) was carried out to produce aromatics using quantitative pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and ZSM-5 zeolite in the hydrogen form was employed as the catalyst. Co-CFP temperature and a parameter called hydrogen to carbon effective ratio (H/C(eff) ratio) were examined for their effects on the relative content of aromatics. Experimental results showed that co-CFP temperature of 600 °C was optimal for the formation of aromatics and other organic pyrolysis products. Besides, H/C(eff) ratio had an important influence on product distribution. The yield of total organic pyrolysis products and relative content of aromatics increased non-linearly with increasing H/C(eff) ratio. There was an apparent synergistic effect between corn stalk and FW during co-CFP process, which promoted the production of aromatics significantly. Co-CFP of biomass and FW was an effective method to produce aromatics and other petrochemicals. Copyright © 2015. Published by Elsevier Ltd.

Lessons from 455 Fusarium polyketide synthases

USDA-ARS?s Scientific Manuscript database

In fungi, polyketide synthases (PKSs) synthesize a structurally diverse array of secondary metabolites (SMs) with a range of biological activities. The most studied SMs are toxic to animals and/or plants, alter plant growth, have beneficial pharmaceutical activities, and/or are brightly colored pigm...

Antimicrobial polyketide furanoterpenoids from seaweed-associated heterotrophic bacterium Bacillus subtilis MTCC 10403.

PubMed

Chakraborty, Kajal; Thilakan, Bini; Raola, Vamshi Krishna

2017-10-01

Brown seaweed Anthophycus longifolius (Turner) Kützing (family Sargassaceae) associated heterotrophic bacterium Bacillus subtilis MTCC 10403 was found to be a potent isolate with broad range of antibacterial activity against important perceptive food pathogens Vibrio parahaemolyticus, V. vulnificus, and Aeromonas hydrophila. This bacterium was positive for polyketide synthetase gene (KC589397), and therefore, was selected to bioprospect specialized metabolites bearing polyketide backbone. Bioactivity-guided chromatographic fractionation of the ethyl acetate extract of the seaweed-associated bacterium segregated four homologous polyketide furanoterpenoids with potential antibacterial activities against clinically important pathogens. The minimum inhibitory concentration (MIC) assay showed that the referral antibiotics tetracycline and ampicillin were active at 25 μg/mL against the test pathogens, whereas the previously undescribed (4E)-methyl 13-((16-(furan-2-yl) ethyl)-octahydro-7-hydroxy-4-((E)-23-methylbut-21-enyl)-2H-chromen-6-yl)-4-methylpent-4-enoate (compound 1) and methyl 3-(hexahydro-9-((E)-3-methylpent-1-enyl)-4H-furo[3,2-g]isochromen-6-yl) propanoate (compound 3) displayed antibacterial activities against the test pathogens at a lesser concentration (MIC < 7 μg/mL). The title compounds were characterized by comprehensive nuclear magnetic resonance and mass spectroscopic experiments. Polyketide synthase catalyzed putative biosynthetic mechanism additionally corroborated the structural ascriptions of the hitherto undescribed furanoterpenoids from seaweed-associated bacterial symbiont. The electronic and hydrophobic parameters appeared to hold a conspicuous part in directing the antibacterial properties of the compounds. Seaweed-associated B. subtilis MTCC 10403 demonstrated to represent a potential source of antimicrobial polyketides for pharmaceutical applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae.

PubMed

Chung, K-R; Ehrenshaft, M; Wetzel, D K; Daub, M E

2003-11-01

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

PubMed

Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

2016-03-01

The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

Type III Polyketide Synthases: Discovery, Characterization, and Engineering

ERIC Educational Resources Information Center

Pitel, Sheryl Beth Rubin

2009-01-01

The polyketides are a diverse group of natural products with important applications in medicine and industry. Industry, especially the pharmaceutical industry, is under pressure to deliver "greener" chemical syntheses that are less environmentally damaging and incorporate renewable resources. There exists potential to replace current…

Convergence of isoprene and polyketide biosynthetic machinery: isoprenyl-S-carrier proteins in the pksX pathway of Bacillus subtilis.

PubMed

Calderone, Christopher T; Kowtoniuk, Walter E; Kelleher, Neil L; Walsh, Christopher T; Dorrestein, Pieter C

2006-06-13

The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide/polyketide secondary metabolite. We used a combination of biochemical and mass spectrometric techniques to assign functional roles to the proteins AcpK, PksC, PksL, PksF, PksG, PksH, and PksI, and we conclude that they act to incorporate an acetate-derived beta-methyl branch on an acetoacetyl-S-carrier protein and ultimately generate a Delta(2)-isoprenyl-S-carrier protein. This work highlights the power of mass spectrometry to elucidate the functions of orphan biosynthetic enzymes, and it details a mechanism by which single-carbon beta-branches can be inserted into polyketide-like structures. This pathway represents a noncanonical route to the construction of prenyl units and serves as a prototype for the intersection of isoprenoid and polyketide biosynthetic manifolds in other natural product biosynthetic pathways.

Kernel based machine learning algorithm for the efficient prediction of type III polyketide synthase family of proteins.

PubMed

Mallika, V; Sivakumar, K C; Jaichand, S; Soniya, E V

2010-07-13

Type III Polyketide synthases (PKS) are family of proteins considered to have significant roles in the biosynthesis of various polyketides in plants, fungi and bacteria. As these proteins shows positive effects to human health, more researches are going on regarding this particular protein. Developing a tool to identify the probability of sequence being a type III polyketide synthase will minimize the time consumption and manpower efforts. In this approach, we have designed and implemented PKSIIIpred, a high performance prediction server for type III PKS where the classifier is Support Vector Machines (SVMs). Based on the limited training dataset, the tool efficiently predicts the type III PKS superfamily of proteins with high sensitivity and specificity. The PKSIIIpred is available at http://type3pks.in/prediction/. We expect that this tool may serve as a useful resource for type III PKS researchers. Currently work is being progressed for further betterment of prediction accuracy by including more sequence features in the training dataset.

Diverse Bacterial PKS Sequences Derived From Okadaic Acid-Producing Dinoflagellates

PubMed Central

Perez, Roberto; Liu, Li; Lopez, Jose; An, Tianying; Rein, Kathleen S.

2008-01-01

Okadaic acid (OA) and the related dinophysistoxins are isolated from dinoflagellates of the genus Prorocentrum and Dinophysis. Bacteria of the Roseobacter group have been associated with okadaic acid producing dinoflagellates and have been previously implicated in OA production. Analysis of 16S rRNA libraries reveals that Roseobacter are the most abundant bacteria associated with OA producing dinoflagellates of the genus Prorocentrum and are not found in association with non-toxic dinoflagellates. While some polyketide synthase (PKS) genes form a highly supported Prorocentrum clade, most appear to be bacterial, but unrelated to Roseobacter or Alpha-Proteobacterial PKSs or those derived from other Alveolates Karenia brevis or Crytosporidium parvum. PMID:18728765

Synthesis of polyketide stereoarrays enabled by a traceless oxonia-Cope rearrangement.

PubMed

Yang, Lin; He, Guoli; Yin, Ruifeng; Zhu, Lili; Wang, Xiaoxia; Hong, Ran

2014-10-20

Polyketide antibiotics bearing skipped polyols represent a synthetic challenge. A SiCl4-promoted oxonia-Cope rearrangement of syn,syn-2-vinyl-1,3-diols was developed to forge an array of 1,5-pentenediols, thus providing versatile motifs for the preparation of 1,2,3,5-stereoarrays in a highly stereoselective manner. Further exploration with Sn(OTf)2 realized the rearrangement of a cross-aldehyde which tactically warrants the utility of the current approach to access complex polyketides. The origin of high stereoselectivity is attributed to a chairlike anti-conformation of the oxonium ion intermediate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemogenomics driven discovery of endogenous polyketide anti-infective compounds from endosymbiotic Emericella variecolor CLB38 and their RNA secondary structure analysis

PubMed Central

Yashavantha Rao, H. C.; Rakshith, Devaraju; Harini, Ballagere Puttaraju; Gurudatt, Doddahosuru Mahadevappa; Satish, Sreedharamurthy

2017-01-01

In the postgenomic era, a new strategy for chemical dereplication of polyketide anti-infective drugs requires novel genomics and chromatographic strategies. An endosymbiotic fungal strain CLB38 was isolated from the root tissue of Combretum latifolium Blume (Combretaceae) which was collected from the Western Ghats of India. The isolate CLB38 was then identified as Emericella variecolor by its characteristic stellate ascospores culture morphology and molecular analysis of ITS nuclear rDNA and intervening 5.8S rRNA gene sequence. ITS2 RNA secondary structure modeling clearly distinguished fungal endosymbiont E. variecolor CLB38 with other lifestyles in the same monophyletic clade. Ethyl acetate fraction of CLB38 explored a broad spectrum of antimicrobial activity against multidrug resistant pathogens. Biosynthetic PKS type-I gene and chromatographic approach afford two polyketide antimicrobial compounds which identified as evariquinone and isoindolones derivative emerimidine A. MIC of purified compounds against test microorganisms ranged between 3.12 μg/ml and 12.5 μg/ml. This research highlights the utility of E. variecolor CLB38 as an anticipate source for anti-infective polyketide metabolites evariquinone and emerimidine A to combat multidrug resistant microorganisms. Here we demonstrates a chemogenomics strategy via the feasibility of PKS type-I gene and chromatographic approach as a proficient method for the rapid prediction and discovery of new polyketides compounds from fungal endosymbionts. PMID:28245269

The Insect Pathogen Serratia marcescens Db10 Uses a Hybrid Non-Ribosomal Peptide Synthetase-Polyketide Synthase to Produce the Antibiotic Althiomycin

PubMed Central

Challis, Gregory L.; Stanley-Wall, Nicola R.; Coulthurst, Sarah J.

2012-01-01

There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1–alb6. Bioinformatic analysis of the proteins encoded by alb1–6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2–Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism. PMID:23028578

The marine sponge-derived polyketide endoperoxide plakortide F acid mediates its antifungal activity by interfering with calcium homeostasis

USDA-ARS?s Scientific Manuscript database

Plakortide F acid (PFA) is a marine-derived polyketide endoperoxide exhibiting strong inhibitory activity against several clinically important fungal pathogens. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Sacch...

Production of New Cladosporin Analogues by Reconstitution of the Polyketide Synthases Responsible for the Biosynthesis of this Antimalarial Agent.

PubMed

Cochrane, Rachel V K; Sanichar, Randy; Lambkin, Gareth R; Reiz, Béla; Xu, Wei; Tang, Yi; Vederas, John C

2016-01-11

The antimalarial agent cladosporin is a nanomolar inhibitor of the Plasmodium falciparum lysyl-tRNA synthetase, and exhibits activity against both blood- and liver-stage infection. Cladosporin can be isolated from the fungus Cladosporium cladosporioides, where it is biosynthesized by a highly reducing (HR) and a non-reducing (NR) iterative type I polyketide synthase (PKS) pair. Genome sequencing of the host organism and subsequent heterologous expression of these enzymes in Saccharomyces cerevisiae produced cladosporin, confirming the identity of the putative gene cluster. Incorporation of a pentaketide intermediate analogue indicated a 5+3 assembly by the HR PKS Cla2 and the NR PKS Cla3 during cladosporin biosynthesis. Advanced-intermediate analogues were synthesized and incorporated by Cla3 to furnish new cladosporin analogues. A putative lysyl-tRNA synthetase resistance gene was identified in the cladosporin gene cluster. Analysis of the active site emphasizes key structural features thought to be important in resistance to cladosporin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli.

PubMed

Menendez-Bravo, Simón; Comba, Santiago; Sabatini, Martín; Arabolaza, Ana; Gramajo, Hugo

2014-07-01

Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Phylogenetic diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff), detection of polyketide synthase gene and their antagonistic activity analysis.

PubMed

Wang, Ya; Gao, Bo Liang; Li, Xi Xi; Zhang, Zhi Bin; Yan, Ri Ming; Yang, Hui Lin; Zhu, Du

2015-11-01

The biodiversity of plant endophytic fungi is enormous, numerous competent endophytic fungi are capable of providing different forms of fitness benefits to host plants and also could produce a wide array of bioactive natural products, which make them a largely unexplored source of novel compounds with potential bioactivity. In this study, we provided a first insights into revealing the diversity of culturable endophytic fungi in Dongxiang wild rice (Oryza rufipogon Griff.) from China using rDNA-ITS phylogenetic analysis. Here, the potential of fungi in producing bioactive natural products was estimated based on the beta-ketosynthase detected in the polyketide synthase (PKS) gene cluster and on the bioassay of antagonistic activity against two rice phytopathogens Thanatephorus cucumeris and Xanthomonas oryzae. A total of 229 endophytic fungal strains were validated in 19 genera. Among the 24 representative strains, 13 strains displayedantagonistic activity against the phytopathogens. Furthermore, PKS genes were detected in 9 strains, indicating their potential for synthesising PKS compounds. Our study confirms the phylogenetic diversity of endophytic fungi in O. rufipogon G. and highlights that endophytic fungi are not only promising resources of biocontrol agents against phytopathogens of rice plants, but also of bioactive natural products and defensive secondary metabolites. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Phosphate Control of Oxytetracycline Production by Streptomyces rimosus Is at the Level of Transcription from Promoters Overlapped by Tandem Repeats Similar to Those of the DNA-Binding Sites of the OmpR Family

PubMed Central

McDowall, Kenneth J.; Thamchaipenet, Arinthip; Hunter, Iain S.

1999-01-01

Physiological studies have shown that Streptomyces rimosus produces the polyketide antibiotic oxytetracycline abundantly when its mycelial growth is limited by phosphate starvation. We show here that transcripts originating from the promoter for one of the biosynthetic genes, otcC (encoding anhydrotetracycline oxygenase), and from a promoter for the divergent otcX genes peak in abundance at the onset of antibiotic production induced by phosphate starvation, indicating that the synthesis of oxytetracycline is controlled, at least in part, at the level of transcription. Furthermore, analysis of the sequences of the promoters for otcC, otcX, and the polyketide synthase (otcY) genes revealed tandem repeats having significant similarity to the DNA-binding sites of ActII-Orf4 and DnrI, which are Streptomyces antibiotic regulatory proteins (SARPs) related to the OmpR family of transcription activators. Together, the above results suggest that oxytetracycline production by S. rimosus requires a SARP-like transcription factor that is either produced or activated or both under conditions of low phosphate concentrations. We also provide evidence consistent with the otrA resistance gene being cotranscribed with otcC as part of a polycistronic message, suggesting a simple mechanism of coordinate regulation which ensures that resistance to the antibiotic increases in proportion to production. PMID:10322002

Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140

PubMed Central

Cheng, Yi-Qiang; Tang, Gong-Li; Shen, Ben

2002-01-01

Leinamycin (LNM), produced by Streptomyces atroolivaceus, is a thiazole-containing hybrid peptide-polyketide natural product structurally characterized with an unprecedented 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a 18-member macrolactam ring. LNM exhibits a broad spectrum of antimicrobial and antitumor activities, most significantly against tumors that are resistant to clinically important anticancer drugs, resulting from its DNA cleavage activity in the presence of a reducing agent. Using a PCR approach to clone a thiazole-forming nonribosomal peptide synthetase (NRPS) as a probe, we localized a 172-kb DNA region from S. atroolivaceus S-140 that harbors the lnm biosynthetic gene cluster. Sequence analysis of 11-kb DNA revealed three genes, lnmG, lnmH, and lnmI, and the deduced product of lnmI is characterized by domains characteristic to both NRPS and polyketide synthase (PKS). The involvement of the cloned gene cluster in LNM biosynthesis was confirmed by disrupting the lnmI gene to generate non-LNM-producing mutants and by characterizing LnmI as a hybrid NRPS-PKS megasynthetase, the NRPS module of which specifies for l-Cys and catalyzes thiazole formation. These results have now set the stage for full investigations of LNM biosynthesis and for generation of novel LNM analogs by combinatorial biosynthesis. PMID:12446651

Heterologous pathway assembly reveals molecular steps of fungal terreic acid biosynthesis.

PubMed

Kong, Chuixing; Huang, Hezhou; Xue, Ying; Liu, Yiqi; Peng, Qiangqiang; Liu, Qi; Xu, Qin; Zhu, Qiaoyun; Yin, Ying; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2018-02-01

Terreic acid is a potential anticancer drug as it inhibits Bruton's tyrosine kinase; however, its biosynthetic molecular steps remain unclear. In this work, the individual reactions of terreic acid biosynthesis were determined by stepwise pathway assembly in a heterologous host, Pichia pastoris, on the basis of previous knockout studies in a native host, Aspergillus terreus. Polyketide synthase AtX was found to catalyze the formation of partially reduced polyketide 6-methylsalicylic acid, followed by 3-methylcatechol synthesis by salicylate 1-monooxygenase AtA-mediated decarboxylative hydroxylation of 6-methylsalicylic acid. Our results show that cytochrome P450 monooxygenase AtE hydroxylates 3-methylcatechol, thus producing the next product, 3-methyl-1,2,4-benzenetriol. A smaller putative cytochrome P450 monooxygenase, AtG, assists with this step. Then, AtD causes epoxidation and hydroxyl oxidation of 3-methyl-1,2,4-benzenetriol and produces a compound terremutin, via which the previously unknown function of AtD was identified as cyclooxygenation. The final step involves an oxidation reaction of a hydroxyl group by a glucose-methanol-choline oxidoreductase, AtC, which leads to the final product: terreic acid. Functions of AtD and AtG were determined for the first time. All the genes were reanalyzed and all intermediates and final products were isolated and identified. Our model fully defines the molecular steps and corrects previous results from the literature.

Comparative Transcriptomics to Identify Novel Genes and Pathways in Dinoflagellates

NASA Astrophysics Data System (ADS)

Ryan, D.

2016-02-01

The unarmored dinoflagellate Karenia brevis is among the most prominent harmful, bloom-forming phytoplankton species in the Gulf of Mexico. During blooms, the polyketides PbTx-1 and PbTx-2 (brevetoxins) are produced by K. brevis. Brevetoxins negatively impact human health and the Gulf shellfish harvest. However, the genes underlying brevetoxin synthesis are currently unknown. Because the K. brevis genome is extremely large ( 1 × 1011 base pairs long), and with a high proportion of repetitive, non-coding DNA, it has not been sequenced. In fact, large, repetitive genomes are common among the dinoflagellate group. High-throughput RNA sequencing technology enabled us to assemble Karenia transcriptomes de novo and investigate potential genes in the brevetoxin pathway through comparative transcriptomics. The brevetoxin profile varies among K. brevis clonal cultures. For example, well-documented Wilson-CCFWC268 typically produces 8-10 pg PbTx per cell, whereas SP1 produces < 2 pg PbTx/cell, and the mutant low-toxin Wilson clone produces undetectable to low (<0.05 pg/cell) amounts. Further, PbTx-2 has been measured in Karenia papilionacea but not Karenia mikimotoi. We compared the transcriptomes of four K. brevis clones (Wilson-CCFWC268, SP3, SP1, and mutant low-toxin Wilson) with K. papilionacea and K. mikimotoi to investigate nucleotide-level genetic variations and differences in gene expression. Of the 85,000 transcripts in the K. brevis transcriptome, 4,600 transcripts, including novel unannotated orthologs and putative polyketide synthases (PKSs), were only expressed by brevetoxin-producing K. brevis and K. papilionacea, not K. mikimotoi. Examination of gene expression between the typical- and low-toxin Wilson clones identified about 3,500 genes with significantly different expression levels, including 2 putative PKSs. One of the 2 PKSs was only found in the brevetoxin-producing Karenia species. These transcriptomes could not have been characterized without high-throughput RNA sequencing.

Metabolic engineering of the shikimate pathway

DOEpatents

Juminaga, Darmawi; Keasling, Jay D.

2017-01-10

The present disclosure relates to engineered microorganisms that produce amino acids and amino acid intermediates. In particular, the disclosure relates to recombinant nucleic acids encoding operons that increase production of aromatic amino acids and the aromatic amino acid intermediate shikimate; microorganisms with increased production of aromatic amino acids and the aromatic amino acid intermediate shikimate; and methods related to the production of aromatic amino acids, the aromatic amino acid intermediate shikimate, and commodity chemicals derived therefrom.

Polyketides from Pestalotiopsis zonata and structure revision of pestalrones A and B.

PubMed

Xu, Xin; Liu, Chang; Dong, Ya-Jing; Liu, Fang-Ru; Xu, Xiu-Mei; Li, De-Sheng; Li, Dan-Yi; Li, Zhan-Lin

2017-11-23

The structures of pestalrones A-B were revised via reinterpretation of the NMR data and a brief chemical transformation from the co-occurring polyketides, in our investigation on the secondary metabolites of Pestalotiopsis zonata, which also afforded a new α-pyrone derivative, pestazonatic acid, and four known analogs.

Functional Reconstitution of a Fungal Natural Product Gene Cluster by Advanced Genome Editing.

PubMed

Weber, Jakob; Valiante, Vito; Nødvig, Christina S; Mattern, Derek J; Slotkowski, Rebecca A; Mortensen, Uffe H; Brakhage, Axel A

2017-01-20

Filamentous fungi produce varieties of natural products even in a strain dependent manner. However, the genetic basis of chemical speciation between strains is still widely unknown. One example is trypacidin, a natural product of the opportunistic human pathogen Aspergillus fumigatus, which is not produced among different isolates. Combining computational analysis with targeted gene editing, we could link a single nucleotide insertion in the polyketide synthase of the trypacidin biosynthetic pathway and reconstitute its production in a nonproducing strain. Thus, we present a CRISPR/Cas9-based tool for advanced molecular genetic studies in filamentous fungi, exploiting selectable markers separated from the edited locus.

Diversity of Secondary Metabolites from Marine Bacillus Species: Chemistry and Biological Activity

PubMed Central

Mondol, Muhammad Abdul Mojid; Shin, Hee Jae; Islam, Mohammad Tofazzal

2013-01-01

Marine Bacillus species produce versatile secondary metabolites including lipopeptides, polypeptides, macrolactones, fatty acids, polyketides, and isocoumarins. These structurally diverse compounds exhibit a wide range of biological activities, such as antimicrobial, anticancer, and antialgal activities. Some marine Bacillus strains can detoxify heavy metals through reduction processes and have the ability to produce carotenoids. The present article reviews the chemistry and biological activities of secondary metabolites from marine isolates. Side by side, the potential for application of these novel natural products from marine Bacillus strains as drugs, pesticides, carotenoids, and tools for the bioremediation of heavy metal toxicity are also discussed. PMID:23941823

Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.

PubMed

Yang, Xinmei; Matsui, Takashi; Kodama, Takeshi; Mori, Takahiro; Zhou, Xiaoxi; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

2016-03-01

In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+β barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-β-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future. Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively. © 2016 Federation of European Biochemical Societies.

Gibepyrone Biosynthesis in the Rice Pathogen Fusarium fujikuroi Is Facilitated by a Small Polyketide Synthase Gene Cluster*

PubMed Central

Janevska, Slavica; Arndt, Birgit; Niehaus, Eva-Maria; Burkhardt, Immo; Rösler, Sarah M.; Brock, Nelson L.; Humpf, Hans-Ulrich; Dickschat, Jeroen S.; Tudzynski, Bettina

2016-01-01

The 2H-pyran-2-one gibepyrone A and its oxidized derivatives gibepyrones B–F have been isolated from the rice pathogenic fungus Fusarium fujikuroi already more than 20 years ago. However, these products have not been linked to the respective biosynthetic genes, and therefore, their biosynthesis has not yet been analyzed on a molecular level. Feeding experiments with isotopically labeled precursors clearly supported a polyketide origin for the formal monoterpenoid gibepyrone A, whereas the terpenoid pathway could be excluded. Targeted gene deletion verified that the F. fujikuroi polyketide synthase PKS13, designated Gpy1, is responsible for gibepyrone A biosynthesis. Next to Gpy1, the ATP-binding cassette transporter Gpy2 is encoded by the gibepyrone gene cluster. Gpy2 was shown to have only a minor impact on the actual efflux of gibepyrone A out of the cell. Instead, we obtained evidence that Gpy2 is involved in gene regulation as it represses GPY1 gene expression. Thus, GPY1 was up-regulated and gibepyrone A production was enhanced both extra- and intracellularly in Δgpy2 mutants. Furthermore, expression of GPY genes is strictly repressed by members of the fungus-specific velvet complex, Vel1, Vel2, and Lae1, whereas Sge1, a major regulator of secondary metabolism in F. fujikuroi, affects gibepyrone biosynthesis in a positive manner. The gibepyrone A derivatives gibepyrones B and D were shown to be produced by cluster-independent P450 monooxygenases, probably to protect the fungus from the toxic product. In contrast, the formation of gibepyrones E and F from gibepyrone A is a spontaneous process and independent of enzymatic activity. PMID:27856636

Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

PubMed Central

Amoutzias, Grigoris D.; Chaliotis, Anargyros; Mossialos, Dimitris

2016-01-01

Considering that 70% of our planet’s surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies are driving the discovery of NRPs and PKs derived from marine microbiomes mainly through two strategies: genome-mining and metagenomics. Microbial genomes are now sequenced at an unprecedented rate and this vast quantity of biological information can be analyzed through genome mining in order to identify gene clusters encoding NRPSes and PKSes of interest. On the other hand, metagenomics is a fast-growing research field which directly studies microbial genomes and their products present in marine environments using culture-independent approaches. The aim of this review is to examine recent developments regarding discovery strategies of bioactive compounds synthesized by NRPS and type-I PKS derived from marine microbiomes and to highlight the vast diversity of NRPSes and PKSes present in marine environments by giving examples of recently discovered bioactive compounds. PMID:27092515

Marine Microbial Secondary Metabolites: Pathways, Evolution and Physiological Roles.

PubMed

Giordano, Daniela; Coppola, Daniela; Russo, Roberta; Denaro, Renata; Giuliano, Laura; Lauro, Federico M; di Prisco, Guido; Verde, Cinzia

2015-01-01

Microbes produce a huge array of secondary metabolites endowed with important ecological functions. These molecules, which can be catalogued as natural products, have long been exploited in medical fields as antibiotics, anticancer and anti-infective agents. Recent years have seen considerable advances in elucidating natural-product biosynthesis and many drugs used today are natural products or natural-product derivatives. The major contribution to recent knowledge came from application of genomics to secondary metabolism and was facilitated by all relevant genes being organised in a contiguous DNA segment known as gene cluster. Clustering of genes regulating biosynthesis in bacteria is virtually universal. Modular gene clusters can be mixed and matched during evolution to generate structural diversity in natural products. Biosynthesis of many natural products requires the participation of complex molecular machines known as polyketide synthases and non-ribosomal peptide synthetases. Discovery of new evolutionary links between the polyketide synthase and fatty acid synthase pathways may help to understand the selective advantages that led to evolution of secondary-metabolite biosynthesis within bacteria. Secondary metabolites confer selective advantages, either as antibiotics or by providing a chemical language that allows communication among species, with other organisms and their environment. Herewith, we discuss these aspects focusing on the most clinically relevant bioactive molecules, the thiotemplated modular systems that include polyketide synthases, non-ribosomal peptide synthetases and fatty acid synthases. We begin by describing the evolutionary and physiological role of marine natural products, their structural/functional features, mechanisms of action and biosynthesis, then turn to genomic and metagenomic approaches, highlighting how the growing body of information on microbial natural products can be used to address fundamental problems in environmental evolution and biotechnology. © 2015 Elsevier Ltd. All rights reserved.

Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity

DOE PAGES

Johnson, Christopher W.; Salvachua, Davinia; Khanna, Payal; ...

2016-04-22

The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces muconate production. A recent study showed that activity of the PCAmore » decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15 g/L. In strains engineered to produce muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92+/-0.48 g/L muconate. Furthermore, this study demonstrates that overcoming the PCA decarboxylase bottleneck can increase muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions.« less

Actinoranone, A Cytotoxic Meroterpenoid of Unprecedented Structure from a Marine Adapted Streptomyces sp

PubMed Central

Nam, Sang-Jip; Kauffman, Christopher A.; Paul, Lauren A.; Jensen, Paul R.

2014-01-01

The isolation and structure elucidation of a new meroterpenoid, actinoranone (1), produced by a marine bacterium closely related to the genus Streptomyces is reported. Actinoranone is composed of an unprecedented dihydronaphthalenone polyketide linked to a bicyclic diterpenoid. The stereochemistry of 1 was defined by application of the advanced Mosher's method and by interpretation of spectroscopic data. Actinoranone (1) is significantly cytotoxic to HCT-116 human colon cancer cells with an LD50 = 2.0 μg/mL. PMID:24152065

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathews, Irimpan I.; Allison, Kim; Robbins, Thomas

The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for sample delivery directly into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes on polyketide synthase reaction dynamics are discussed.

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

DOE PAGES

Mathews, Irimpan I.; Allison, Kim; Robbins, Thomas; ...

2017-08-23

The crystal structure of the trans-acyltransferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for sample delivery directly into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes on polyketide synthase reaction dynamics are discussed.

The Polyketide Components of Waxes and the Cer-cqu Gene Cluster Encoding a Novel Polyketide Synthase, the β-Diketone Synthase, DKS.

PubMed

von Wettstein-Knowles, Penny

2017-07-10

The primary function of the outermost, lipophilic layer of plant aerial surfaces, called the cuticle, is preventing non-stomatal water loss. Its exterior surface is often decorated with wax crystals, imparting a blue-grey color. Identification of the barley Cer-c , -q and -u genes forming the 101 kb Cer-cqu gene cluster encoding a novel polyketide synthase-the β-diketone synthase (DKS), a lipase/carboxyl transferase, and a P450 hydroxylase, respectively, establishes a new, major pathway for the synthesis of plant waxes. The major product is a β-diketone (14,16-hentriacontane) aliphatic that forms long, thin crystalline tubes. A pathway branch leads to the formation of esterified alkan-2-ols.

DHA Production in Escherichia coli by Expressing Reconstituted Key Genes of Polyketide Synthase Pathway from Marine Bacteria.

PubMed

Peng, Yun-Feng; Chen, Wen-Chao; Xiao, Kang; Xu, Lin; Wang, Lian; Wan, Xia

2016-01-01

The gene encoding phosphopantetheinyl transferase (PPTase), pfaE, a component of the polyketide synthase (PKS) pathway, is crucial for the production of docosahexaenoic acid (DHA, 22:6ω3), along with the other pfa cluster members pfaA, pfaB, pfaC and pfaD. DHA was produced in Escherichia coli by co-expressing pfaABCD from DHA-producing Colwellia psychrerythraea 34H with one of four pfaE genes from bacteria producing arachidonic acid (ARA, 20:4ω6), eicosapentaenoic acid (EPA, 20:5ω3) or DHA, respectively. Substitution of the pfaE gene from different strain source in E. coli did not influence the function of the PKS pathway producing DHA, although they led to different DHA yields and fatty acid profiles. This result suggested that the pfaE gene could be switchable between these strains for the production of DHA. The DHA production by expressing the reconstituted PKS pathway was also investigated in different E. coli strains, at different temperatures, or with the treatment of cerulenin. The highest DHA production, 2.2 mg of DHA per gram of dry cell weight or 4.1% of total fatty acids, was obtained by co-expressing pfaE(EPA) from the EPA-producing strain Shewanella baltica with pfaABCD in DH5α. Incubation at low temperature (10-15°C) resulted in higher accumulation of DHA compared to higher temperatures. The addition of cerulenin to the medium increased the proportion of DHA and saturated fatty acids, including C12:0, C14:0 and C16:0, at the expense of monounsaturated fatty acids, including C16:1 and C18:1. Supplementation with 1 mg/L cerulenin resulted in the highest DHA yield of 2.4 mg/L upon co-expression of pfaE(DHA) from C. psychrerythraea.

UV irradiation of polycyclic aromatic hydrocarbons in ices: production of alcohols, quinones, and ethers

NASA Technical Reports Server (NTRS)

Bernstein, M. P.; Sandford, S. A.; Allamandola, L. J.; Gillette, J. S.; Clemett, S. J.; Zare, R. N.

1999-01-01

Polycyclic aromatic hydrocarbons (PAHs) in water ice were exposed to ultraviolet (UV) radiation under astrophysical conditions, and the products were analyzed by infrared spectroscopy and mass spectrometry. Peripheral carbon atoms were oxidized, producing aromatic alcohols, ketones, and ethers, and reduced, producing partially hydrogenated aromatic hydrocarbons, molecules that account for the interstellar 3.4-micrometer emission feature. These classes of compounds are all present in carbonaceous meteorites. Hydrogen and deuterium atoms exchange readily between the PAHs and the ice, which may explain the deuterium enrichments found in certain meteoritic molecules. This work has important implications for extraterrestrial organics in biogenesis.

Synthesis of Renewable meta-Xylylenediamine from Biomass-Derived Furfural.

PubMed

Scodeller, Ivan; Mansouri, Samir; Morvan, Didier; Muller, Eric; de Oliveira Vigier, Karine; Wischert, Raphael; Jérôme, François

2018-04-30

We report the synthesis of biomass-derived functionalized aromatic chemicals from furfural, a building block nowadays available in large scale from low-cost biomass. The scientific strategy relies on a Diels-Alder/aromatization sequence. By controlling the rate of each step, it was possible to produce exclusively the meta aromatic isomer. In particular, through this route, we describe the synthesis of renewably sourced meta-xylylenediamine (MXD). Transposition of this work to other furfural-derived chemicals is also discussed and reveals that functionalized biomass-derived aromatics (benzaldehyde, benzylamine, etc.) can be potentially produced, according to this route. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacterial fermentation platform for producing artificial aromatic amines

PubMed Central

Masuo, Shunsuke; Zhou, Shengmin; Kaneko, Tatsuo; Takaya, Naoki

2016-01-01

Aromatic amines containing an aminobenzene or an aniline moiety comprise versatile natural and artificial compounds including bioactive molecules and resources for advanced materials. However, a bio-production platform has not been implemented. Here we constructed a bacterial platform for para-substituted aminobenzene relatives of aromatic amines via enzymes in an alternate shikimate pathway predicted in a Pseudomonad bacterium. Optimization of the metabolic pathway in Escherichia coli cells converted biomass glucose to 4-aminophenylalanine with high efficiency (4.4 g L−1 in fed-batch cultivation). We designed and produced artificial pathways that mimicked the fungal Ehrlich pathway in E. coli and converted 4-aminophenylalanine into 4-aminophenylethanol and 4-aminophenylacetate at 90% molar yields. Combining these conversion systems or fungal phenylalanine decarboxylases, the 4-aminophenylalanine-producing platform fermented glucose to 4-aminophenylethanol, 4-aminophenylacetate, and 4-phenylethylamine. This original bacterial platform for producing artificial aromatic amines highlights their potential as heteroatoms containing bio-based materials that can replace those derived from petroleum. PMID:27167511

Metabolic Interplay between the Asian Citrus Psyllid and Its Profftella Symbiont: An Achilles’ Heel of the Citrus Greening Insect Vector

PubMed Central

Ramsey, John S.; Johnson, Richard S.; Hoki, Jason S.; Kruse, Angela; Mahoney, Jaclyn; Hilf, Mark E.; Hunter, Wayne B.; Hall, David G.; Schroeder, Frank C.; MacCoss, Michael J.; Cilia, Michelle

2015-01-01

‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including ‘Candidatus Profftella armatura’, are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide. PMID:26580079

SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone.

PubMed

Schäfer, Martin; Le, Tung B K; Hearnshaw, Stephen J; Maxwell, Anthony; Challis, Gregory L; Wilkinson, Barrie; Buttner, Mark J

2015-06-19

Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone. Copyright © 2015. Published by Elsevier Ltd.

Production of aromatics from di- and polyoxygenates

DOEpatents

Beck, Taylor; Blank, Brian; Jones, Casey; Woods, Elizabeth; Cortright, Randy

2016-08-02

Methods, catalysts, and reactor systems for producing in high yield aromatic chemicals and liquid fuels from a mixture of oxygenates comprising di- and polyoxygenates are disclosed. Also disclosed are methods, catalysts, and reactor systems for producing aromatic chemicals and liquid fuels from oxygenated hydrocarbons such as carbohydrates, sugars, sugar alcohols, sugar degradation products, and the like; and methods, catalysts, and reactor systems for producing the mixture of oxygenates from oxygenated hydrocarbons such as carbohydrates, sugars, sugar alcohols, sugar degradation products, and the like. The disclosed catalysts for preparing the mixture of oxygenates comprise a Ni.sub.nSn.sub.m alloy and a crystalline alumina support.

Production of aromatics from di- and polyoxygenates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beck, Taylor; Blank, Brian; Jones, Casey

Methods, catalysts, and reactor systems for producing in high yield aromatic chemicals and liquid fuels from a mixture of oxygenates comprising di- and polyoxygenates are disclosed. Also disclosed are methods, catalysts, and reactor systems for producing aromatic chemicals and liquid fuels from oxygenated hydrocarbons such as carbohydrates, sugars, sugar alcohols, sugar degradation products, and the like; and methods, catalysts, and reactor systems for producing the mixture of oxygenates from oxygenated hydrocarbons such as carbohydrates, sugars, sugar alcohols, sugar degradation products, and the like. The disclosed catalysts for preparing the mixture of oxygenates comprise a Ni.sub.nSn.sub.m alloy and a crystalline aluminamore » support.« less

Production of aromatics from di- and polyoxygenates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beck, Taylor; Blank, Brian; Jones, Casey

Methods, catalysts, and reactor systems for producing in high yield aromatic chemicals and liquid fuels from a mixture of oxygenates comprising di- and polyoxygenates are disclosed. Also disclosed are methods, catalysts, and reactor systems for producing aromatic chemicals and liquid fuels from oxygenated hydrocarbons such as carbohydrates, sugars, sugar alcohols, sugar degradation products, and the like; and methods, catalysts, and reactor systems for producing the mixture of oxygenates from oxygenated hydrocarbons such as carbohydrates, sugars, sugar alcohols, sugar degradation products, and the like. The disclosed catalysts for preparing the mixture of oxygenates comprise a Group VIII metal and a crystallinemore » alumina support.« less

The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography

PubMed Central

Allison, Kim; Robbins, Thomas; Lyubimov, Artem Y.; Uervirojnangkoorn, Monarin; Brunger, Axel T.; Khosla, Chaitan; DeMirci, Hasan; McPhillips, Scott E.; Hollenbeck, Michael; Soltis, Michael; Cohen, Aina E.

2017-01-01

The crystal structure of the trans-acyltrans-ferase (AT) from the disorazole polyketide synthase (PKS) was determined at room temperature to a resolution of 2.5 Å using a new method for the direct delivery of the sample into an X-ray free-electron laser. A novel sample extractor efficiently delivered limited quantities of microcrystals directly from the native crystallization solution into the X-ray beam at room temperature. The AT structure revealed important catalytic features of this core PKS enzyme, including the occurrence of conformational changes around the active site. The implications of these conformational changes for polyketide synthase reaction dynamics are discussed. PMID:28832129

The Polyketide Components of Waxes and the Cer-cqu Gene Cluster Encoding a Novel Polyketide Synthase, the β-Diketone Synthase, DKS

PubMed Central

von Wettstein-Knowles, Penny

2017-01-01

The primary function of the outermost, lipophilic layer of plant aerial surfaces, called the cuticle, is preventing non-stomatal water loss. Its exterior surface is often decorated with wax crystals, imparting a blue–grey color. Identification of the barley Cer-c, -q and -u genes forming the 101 kb Cer-cqu gene cluster encoding a novel polyketide synthase—the β-diketone synthase (DKS), a lipase/carboxyl transferase, and a P450 hydroxylase, respectively, establishes a new, major pathway for the synthesis of plant waxes. The major product is a β-diketone (14,16-hentriacontane) aliphatic that forms long, thin crystalline tubes. A pathway branch leads to the formation of esterified alkan-2-ols. PMID:28698520

Biological effects of paenilamicin, a secondary metabolite antibiotic produced by the honey bee pathogenic bacterium Paenibacillus larvae.

PubMed

Garcia-Gonzalez, Eva; Müller, Sebastian; Hertlein, Gillian; Heid, Nina; Süssmuth, Roderich D; Genersch, Elke

2014-10-01

Paenibacillus larvae is the etiological agent of American Foulbrood (AFB) a world-wide distributed devastating disease of the honey bee brood. Previous comparative genome analysis and more recently, the elucidation of the bacterial genome, provided evidence that this bacterium harbors putative functional nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) and therefore, might produce nonribosomal peptides (NRPs) and polyketides (PKs). Such biosynthesis products have been shown to display a wide-range of biological activities such as antibacterial, antifungal or cytotoxic activity. Herein we present an in silico analysis of the first NRPS/PKS hybrid of P. larvae and we show the involvement of this cluster in the production of a compound named paenilamicin (Pam). For the characterization of its in vitro and in vivo bioactivity, a knock-out mutant strain lacking the production of Pam was constructed and subsequently compared to wild-type species. This led to the identification of Pam by mass spectrometry. Purified Pam-fractions showed not only antibacterial but also antifungal and cytotoxic activities. The latter suggested a direct effect of Pam on honey bee larval death which could, however, not be corroborated in laboratory infection assays. Bee larvae infected with the non-producing Pam strain showed no decrease in larval mortality, but a delay in the onset of larval death. We propose that Pam, although not essential for larval mortality, is a virulence factor of P. larvae influencing the time course of disease. These findings are not only of significance in elucidating and understanding host-pathogen interactions but also within the context of the quest for new compounds with antibiotic activity for drug development. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Development of genetically engineered bacteria for production of selected aromatic compounds

DOEpatents

Ward, Thomas E.; Watkins, Carolyn S.; Bulmer, Deborah K.; Johnson, Bruce F.; Amaratunga, Mohan

2001-01-01

The cloning and expression of genes in the common aromatic pathway of E. coli are described. A compound for which chorismate, the final product of the common aromatic pathway, is an anabolic intermediate can be produced by cloning and expressing selected genes of the common aromatic pathway and the genes coding for enzymes necessary to convert chorismate to the selected compound. Plasmids carrying selected genes of the common aromatic pathway are also described.

Genetic diversity, morphological uniformity and polyketide production in dinoflagellates (Amphidinium, Dinoflagellata).

PubMed

Murray, Shauna A; Garby, Tamsyn; Hoppenrath, Mona; Neilan, Brett A

2012-01-01

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a 'model' dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of 'cryptic' species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them.

Genetic Diversity, Morphological Uniformity and Polyketide Production in Dinoflagellates (Amphidinium, Dinoflagellata)

PubMed Central

Hoppenrath, Mona; Neilan, Brett A.

2012-01-01

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a ‘model’ dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of ‘cryptic’ species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them. PMID:22675531

Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization.

PubMed

Lowell, Andrew N; DeMars, Matthew D; Slocum, Samuel T; Yu, Fengan; Anand, Krithika; Chemler, Joseph A; Korakavi, Nisha; Priessnitz, Jennifer K; Park, Sung Ryeol; Koch, Aaron A; Schultz, Pamela J; Sherman, David H

2017-06-14

Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.

Molecular architectures of benzoic acid-specific type III polyketide synthases

PubMed Central

Stewart, Charles; Woods, Kate; Macias, Greg; Allan, Andrew C.; Noel, Joseph P.

2017-01-01

Biphenyl synthase and benzophenone synthase constitute an evolutionarily distinct clade of type III polyketide synthases (PKSs) that use benzoic acid-derived substrates to produce defense metabolites in plants. The use of benzoyl-CoA as an endogenous substrate is unusual for type III PKSs. Moreover, sequence analyses indicate that the residues responsible for the functional diversification of type III PKSs are mutated in benzoic acid-specific type III PKSs. In order to gain a better understanding of structure–function relationships within the type III PKS family, the crystal structures of biphenyl synthase from Malus × domestica and benzophenone synthase from Hypericum androsaemum were compared with the structure of an archetypal type III PKS: chalcone synthase from Malus × domestica. Both biphenyl synthase and benzophenone synthase contain mutations that reshape their active-site cavities to prevent the binding of 4-coumaroyl-CoA and to favor the binding of small hydrophobic substrates. The active-site cavities of biphenyl synthase and benzophenone synthase also contain a novel pocket associated with their chain-elongation and cyclization reactions. Collectively, these results illuminate structural determinants of benzoic acid-specific type III PKSs and expand the understanding of the evolution of specialized metabolic pathways in plants. PMID:29199980

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases*

PubMed Central

Klaus, Maja; Ostrowski, Matthew P.; Austerjost, Jonas; Robbins, Thomas; Lowry, Brian; Cane, David E.; Khosla, Chaitan

2016-01-01

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs. PMID:27246853

An In Planta-Expressed Polyketide Synthase Produces (R)-Mellein in the Wheat Pathogen Parastagonospora nodorum

PubMed Central

Krill, Christian; Barrow, Russell A.; Chen, Shasha; Trengove, Robert; Oliver, Richard P.; Solomon, Peter S.

2014-01-01

Parastagonospora nodorum is a pathogen of wheat that affects yields globally. Previous transcriptional analysis identified a partially reducing polyketide synthase (PR-PKS) gene, SNOG_00477 (SN477), in P. nodorum that is highly upregulated during infection of wheat leaves. Disruption of the corresponding SN477 gene resulted in the loss of production of two compounds, which we identified as (R)-mellein and (R)-O-methylmellein. Using a Saccharomyces cerevisiae yeast heterologous expression system, we successfully demonstrated that SN477 is the only enzyme required for the production of (R)-mellein. This is the first identification of a fungal PKS that is responsible for the synthesis of (R)-mellein. The P. nodorum ΔSN477 mutant did not show any significant difference from the wild-type strain in its virulence against wheat. However, (R)-mellein at 200 μg/ml inhibited the germination of wheat (Triticum aestivum) and barrel medic (Medicago truncatula) seeds. Comparative sequence analysis identified the presence of mellein synthase (MLNS) homologues in several Dothideomycetes and two sodariomycete genera. Phylogenetic analysis suggests that the MLNSs in fungi and bacteria evolved convergently from fungal and bacterial 6-methylsalicylic acid synthases. PMID:25326302

Interkingdom Gene Transfer of a Hybrid NPS/PKS from Bacteria to Filamentous Ascomycota

PubMed Central

Lawrence, Daniel P.; Kroken, Scott; Pryor, Barry M.; Arnold, A. Elizabeth

2011-01-01

Nonribosomal peptides (NRPs) and polyketides (PKs) are ecologically important secondary metabolites produced by bacteria and fungi using multidomain enzymes called nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively. Previous phylogenetic analyses of fungal NRPSs and PKSs have suggested that a few of these genes were acquired by fungi via horizontal gene transfer (HGT) from bacteria, including a hybrid NPS/PKS found in Cochliobolus heterostrophus (Dothideomycetes, Ascomycota). Here, we identify this hybrid gene in fungi representing two additional classes of Ascomycota (Aspergillus spp., Microsporum canis, Arthroderma spp., and Trichophyton spp., Eurotiomycetes; Chaetomium spp. and Metarhizium spp., Sordariomycetes) and use phylogenetic analyses of the most highly conserved domains from NRPSs (adenylation (A) domain) and PKSs (ketoacyl synthase (KS) domain) to examine the hypothesis that the hybrid NPS7/PKS24 was acquired by fungi from bacteria via HGT relatively early in the evolution of the Pezizomycotina. Our results reveal a unique ancestry of the A domain and KS domain in the hybrid gene relative to known fungal NRPSs and PKSs, provide strong evidence for HGT of the hybrid gene from a putative bacterial donor in the Burkholderiales, and suggest the HGT event occurred early in the evolution of the filamentous Ascomycota. PMID:22140558

Novel and Efficient Synthesis of the Promising Drug Candidate Discodermolide

DTIC Science & Technology

2010-02-01

stereotriad building blocks for discodermolide and related polyketide antibiotics could be obtained from variations on a short, scalable scheme that did...chains required for the chemical synthesis of the nonaromatic polyketides is usually based on the iterative lengthening of an acyclic substituted chain...burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense

Polyketide mimetics yield structural and mechanistic insights into product template domain function in nonreducing polyketide synthases

PubMed Central

Barajas, Jesus F.; Shakya, Gaurav; Moreno, Gabriel; Rivera, Heriberto; Jackson, David R.; Topper, Caitlyn L.; Vagstad, Anna L.; La Clair, James J.; Townsend, Craig A.; Burkart, Michael D.; Tsai, Shiou-Chuan

2017-01-01

Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-β-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-β-ketones for in vitro studies. We describe here the crystallographic application of “atom replacement” mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-β-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4′-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and the role of a protein-coordinated water network to selectively activate the C9 carbonyl for nucleophilic addition. The importance of the 4′-phosphate at the distal end of the pantetheine arm is demonstrated to both facilitate delivery of the heptaketide mimetic deep into the PT active site and anchor one end of this linear array to precisely meter C4 into close proximity to the catalytic His1345. Additional structural features, docking simulations, and mutational experiments characterize protein–substrate mimic interactions, which likely play roles in orienting and stabilizing interactions during the native multistep catalytic cycle. These findings afford a view of a polyketide “atom-replaced” mimetic in a NR-PKS active site that could prove general for other PKS domains. PMID:28484029

Unexpected link between polyketide synthase and calcium carbonate biomineralization.

PubMed

Hojo, Motoki; Omi, Ai; Hamanaka, Gen; Shindo, Kazutoshi; Shimada, Atsuko; Kondo, Mariko; Narita, Takanori; Kiyomoto, Masato; Katsuyama, Yohei; Ohnishi, Yasuo; Irie, Naoki; Takeda, Hiroyuki

2015-01-01

Calcium carbonate biominerals participate in diverse physiological functions. Despite intensive studies, little is known about how mineralization is initiated in organisms. We analyzed the medaka spontaneous mutant, ha, defective in otolith (calcareous ear stone) formation. ha lacks a trigger for otolith mineralization, and the causative gene was found to encode polyketide synthase (pks), a multifunctional enzyme mainly found in bacteria, fungi, and plant. Subsequent experiments demonstrate that the products of medaka PKS, most likely polyketides or their derivatives, act as nucleation facilitators in otolith mineralization. The generality of this novel PKS function is supported by the essential role of echinoderm PKS in calcareous skeleton formation together with the presence of PKSs in a much wider range of animals from coral to vertebrates. The present study first links PKS to biomineralization and provides a genetic cue for biogeochemistry of carbon and calcium cycles.

ClusterMine360: a database of microbial PKS/NRPS biosynthesis

PubMed Central

Conway, Kyle R.; Boddy, Christopher N.

2013-01-01

ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and non-ribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has >200 gene clusters from >185 compound families. It also features a unique sequence repository containing >10 000 polyketide synthase/non-ribosomal peptide synthetase domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. We are confident that this database will be a useful resource for members of the polyketide synthases/non-ribosomal peptide synthetases research community, enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information. PMID:23104377

Genomic mutational analysis of the impact of the classical strain improvement program on β-lactam producing Penicillium chrysogenum.

PubMed

Salo, Oleksandr V; Ries, Marco; Medema, Marnix H; Lankhorst, Peter P; Vreeken, Rob J; Bovenberg, Roel A L; Driessen, Arnold J M

2015-11-14

Penicillium chrysogenum is a filamentous fungus that is employed as an industrial producer of β-lactams. The high β-lactam titers of current strains is the result of a classical strain improvement program (CSI) starting with a wild-type like strain more than six decades ago. This involved extensive mutagenesis and strain selection for improved β-lactam titers and growth characteristics. However, the impact of the CSI on the secondary metabolism in general remains unknown. To examine the impact of CSI on secondary metabolism, a comparative genomic analysis of β-lactam producing strains was carried out by genome sequencing of three P. chrysogenum strains that are part of a lineage of the CSI, i.e., strains NRRL1951, Wisconsin 54-1255, DS17690, and the derived penicillin biosynthesis cluster free strain DS68530. CSI has resulted in a wide spread of mutations, that statistically did not result in an over- or underrepresentation of specific gene classes. However, in this set of mutations, 8 out of 31 secondary metabolite genes (20 polyketide synthases and 11 non-ribosomal peptide synthetases) were targeted with a corresponding and progressive loss in the production of a range of secondary metabolites unrelated to β-lactam production. Additionally, key Velvet complex proteins (LeaA and VelA) involved in global regulation of secondary metabolism have been repeatedly targeted for mutagenesis during CSI. Using comparative metabolic profiling, the polyketide synthetase gene cluster was identified that is responsible for sorbicillinoid biosynthesis, a group of yellow-colored metabolites that are abundantly produced by early production strains of P. chrysogenum. The classical industrial strain improvement of P. chrysogenum has had a broad mutagenic impact on metabolism and has resulted in silencing of specific secondary metabolite genes with the concomitant diversion of metabolism towards the production of β-lactams.

Crystallization and preliminary X-ray diffraction studies of polyketide synthase-1 (PKS-1) from Cannabis sativa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taguchi, Chiho; Quantum Beam Science Directorate, Japan Atomic Energy Agency; Taura, Futoshi

Polyketide synthase-1 from C. sativa has been crystallized. The crystal diffracted to 1.55 Å resolution with sufficient quality for further structure determination. Polyketide synthase-1 (PKS-1) is a novel type III polyketide synthase that catalyzes the biosynthesis of hexanoyl triacetic acid lactone in Cannabis sativa (Mexican strain). PKS-1 was overproduced in Escherichia coli, purified and finally crystallized in two different space groups. The crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M calcium acetate and 20%(w/v) polyethylene glycol 3350 diffracted to 1.65 Å resolution and belonged to space group P1, with unit-cell parameters a = 54.3, b =more » 59.3, c = 62.6 Å, α = 69, β = 81, γ = 80°. Another crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M sodium chloride and 20%(w/v) polyethylene glycol 3350 diffracted to 1.55 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 54.3, b = 110, c = 130 Å. These data will enable us to determine the crystal structure of PKS-1.« less

Detection of azo dyes and aromatic amines in women under garment

PubMed Central

NGUYEN, THAO; SALEH, MAHMOUD A.

2016-01-01

Women are exposed to several chemical additives including azo dyes that exist in textile materials that are a potential health hazard for consumers. Our objective was to analyze suspected carcinogenic azo dyes and their degradation aromatic amines in women's panties underwear using a fast and simple method for quantification. Here, we evaluated 120 different samples of women underwear for their potential release of aromatic amines to the skin. Seventy four samples yielded low level mixtures of aromatic amines; however eighteen samples were found to produce greater than 200 mg/kg (ppm) of aromatic amines. Azo dyes in these 18 samples were extracted from the fabrics and analyzed by reverse phase thin layer chromatography in tandem with atmospheric pressure chemical ionization mass spectrometry. Eleven azo dyes were identified based on their mass spectral data and the chemical structure of the aromatic amine produced from these samples. We demonstrate that planar chromatography and mass spectrometry can be really helpful in confirming the identity of the azo dyes, offering highly relevant molecular information of the responsible compounds in the fabrics. With the growing concern about the consumer goods, analysis of aromatic amines in garments has become a highly important issue. PMID:27149414

Process for lowering the dielectric constant of polyimides using diamic acid additives

NASA Technical Reports Server (NTRS)

Stoakley, Diane M. (Inventor); St.clair, Anne K. (Inventor)

1990-01-01

Linear aromatic polyimides with low dielectric constants are produced by adding a diamic acid additive to the polyamic acid resin formed by the condensation of an aromatic dianhydride with an aromatic diamine. The resulting modified polyimide is a better electrical insulator than state-of-the-art commercially available polyimides.

Curvularides A-E: antifungal hybrid peptide-polyketides from the endophytic fungus Curvularia geniculata.

PubMed

Chomcheon, Porntep; Wiyakrutta, Suthep; Aree, Thammarat; Sriubolmas, Nongluksna; Ngamrojanavanich, Nattaya; Mahidol, Chulabhorn; Ruchirawat, Somsak; Kittakoop, Prasat

2010-09-24

Five new hybrid peptide-polyketides, curvularides A-E (1-5), were isolated from the endophytic fungus Curvularia geniculata, which was obtained from the limbs of Catunaregam tomentosa. Structure elucidation for curvularides A-E (1-5) was accomplished by analysis of spectroscopic data, as well as by single-crystal X-ray crystallography. Curvularide B (2) exhibited antifungal activity against Candida albicans, and it also showed synergistic activity with a fluconazole drug.

Allelopathic Polyketides from an Endolichenic Fungus Myxotrichum SP. by Using OSMAC Strategy.

PubMed

Yuan, Chao; Guo, Yu-Hua; Wang, Hai-Ying; Ma, Xiao-Jun; Jiang, Tao; Zhao, Jun-Ling; Zou, Zhong-Mei; Ding, Gang

2016-02-03

Three new polyketides myxotritones A-C (2-4), together with a new natural product 7,8-dihydro-7R,8S-dihydroxy-3,7-dimethyl-2-benzopyran-6-one (1) were obtained from the endolichenic fungus Myxotrichum sp. by using OMSAC (One Strain, Many Compounds) method. The planar structures of these new compounds were determined by NMR experiment and HRESIMS data, and the absolute configuration of 1 was established by X-ray diffraction, and the stereochemistry of the new compounds 2-4 were determined by same biosynthesis origin, and similar CD spectra with 1. Allelopathic test showed that compound 4 significantly retarded root elongation of Arabidopsis thaliana seed, indicating that this fungus might contribute to the defense of its host lichen. From the view of biosynthetic pathway, all four compounds 1-4 might be originated from Non-Reduced Polyketide synthase (NR-PKS).

E. coli and colorectal cancer: a complex relationship that deserves a critical mindset.

PubMed

Wassenaar, Trudy M

2018-06-17

To the multiple factors that may eventually result in colorectal cancer (CRC), strains of E. coli have now been added, in particular strains producing colibactin from their polyketide synthesis (pks) locus. The evidence and mechanistic explanations for this unfortunate effect of what is in most cases a harmless commensal are discussed in the first part of this review. In the second part, observations are presented and discussed that do not fit with the hypothesis that colibactin-producing E. coli produce CRC. The last part of this review is reserved for an alternative explanation of the function of this enigmatic colibactin, a toxin that has not yet been isolated. It is hypothesized that E. coli preferentially colonizes cancerous lesions as an effect rather than a cause and that colibactin production provides a selective advantage to compete with other bacteria.

The chemistry and beneficial bioactivities of carvacrol (4-isopropyl-2-methylphenol), a component of essential oils produced by aromatic plants and spices

USDA-ARS?s Scientific Manuscript database

Aromatic plants produce organic compounds that may be involved in the defense of plants against phytopathogenic insects, bacteria, fungi, and viruses. One of these compounds called carvacrol that is found in high concentrations in essential oils such as oregano has been reported to exhibit numerous...

Evidence for the Biosynthesis of Bryostatins by the Bacterial Symbiont “Candidatus Endobugula sertula” of the Bryozoan Bugula neritina

PubMed Central

Davidson, S. K.; Allen, S. W.; Lim, G. E.; Anderson, C. M.; Haygood, M. G.

2001-01-01

The marine bryozoan, Bugula neritina, is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont “Candidatus Endobugula sertula.” In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-“E. sertula” association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from B. neritina directly and use these to produce bryostatins in heterologous host bacteria. PMID:11571152

A combined approach of classical mutagenesis and rational metabolic engineering improves rapamycin biosynthesis and provides insights into methylmalonyl-CoA precursor supply pathway in Streptomyces hygroscopicus ATCC 29253.

PubMed

Jung, Won Seok; Yoo, Young Ji; Park, Je Won; Park, Sung Ryeol; Han, Ah Reum; Ban, Yeon Hee; Kim, Eun Ji; Kim, Eunji; Yoon, Yeo Joon

2011-09-01

Rapamycin is a macrocyclic polyketide with immunosuppressive, antifungal, and anticancer activity produced by Streptomyces hygroscopicus ATCC 29253. Rapamycin production by a mutant strain (UV2-2) induced by ultraviolet mutagenesis was improved by approximately 3.2-fold (23.6 mg/l) compared to that of the wild-type strain. The comparative analyses of gene expression and intracellular acyl-CoA pools between wild-type and the UV2-2 strains revealed that the increased production of rapamycin in UV2-2 was due to the prolonged expression of rapamycin biosynthetic genes, but a depletion of intracellular methylmalonyl-CoA limited the rapamycin biosynthesis of the UV2-2 strain. Therefore, three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of rapamycin: propionyl-CoA carboxylase (PCC), methylmalonyl-CoA mutase, and methylmalonyl-CoA ligase. Among them, only the PCC pathway along with supplementation of propionate was found to be effective for an increase in intracellular pool of methylmalonyl-CoA and rapamycin titers in UV2-2 strain (42.8 mg/l), indicating that the PCC pathway is a major methylmalonyl-CoA supply pathway in the rapamycin producer. These results demonstrated that the combined approach involving traditional mutagenesis and metabolic engineering could be successfully applied to the diagnosis of yield-limiting factors and the enhanced production of industrially and clinically important polyketide compounds.

Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

PubMed Central

Horsman, Geoff P.; Chen, Yihua; Thorson, Jon S.; Shen, Ben

2010-01-01

Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: (i) all five cognate PKSE-TE pairs in Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence. PMID:20534556

Synthesis of condensed phases containing polycyclic aromatic hydrocarbons fullerenes and nanotubes

DOEpatents

Reilly, Peter T. A.

2004-10-19

The invention relates to methods for producing polycyclic aromatic hydrocarbons, fullerenes, and nanotubes, comprising: a. heating at least one carbon-containing material to form a condensed phase comprising at least one polycyclic aromatic hydrocarbon; b. collecting at least some of the condensed phase; c. reacting the condensed phase to form fullerenes and/or nanotubes.

Steps towards the synthetic biology of polyketide biosynthesis.

PubMed

Cummings, Matthew; Breitling, Rainer; Takano, Eriko

2014-02-01

Nature is providing a bountiful pool of valuable secondary metabolites, many of which possess therapeutic properties. However, the discovery of new bioactive secondary metabolites is slowing down, at a time when the rise of multidrug-resistant pathogens and the realization of acute and long-term side effects of widely used drugs lead to an urgent need for new therapeutic agents. Approaches such as synthetic biology are promising to deliver a much-needed boost to secondary metabolite drug development through plug-and-play optimized hosts and refactoring novel or cryptic bacterial gene clusters. Here, we discuss this prospect focusing on one comprehensively studied class of clinically relevant bioactive molecules, the polyketides. Extensive efforts towards optimization and derivatization of compounds via combinatorial biosynthesis and classical engineering have elucidated the modularity, flexibility and promiscuity of polyketide biosynthetic enzymes. Hence, a synthetic biology approach can build upon a solid basis of guidelines and principles, while providing a new perspective towards the discovery and generation of novel and new-to-nature compounds. We discuss the lessons learned from the classical engineering of polyketide synthases and indicate their importance when attempting to engineer biosynthetic pathways using synthetic biology approaches for the introduction of novelty and overexpression of products in a controllable manner. © 2013 The Authors FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

Isolation of polyketides from Prymnesium parvum (Haptophyta) and their detection by liquid chromatography/mass spectrometry metabolic fingerprint analysis.

PubMed

Manning, Schonna R; La Claire Ii, John W

2013-11-15

Prymnesium parvum is a microalga that forms blooms coupled with the presence of potent exotoxins; however, no chemical standards are currently available for the toxins. Streamlined methods are presented for the separation and enrichment of polyketide toxins, prymnesin-1 (prym1) and prymnesin-2 (prym2). Prymnesins were separated by reversed-phase chromatography and detected by positive-mode electrospray ionization MS to generate a unique metabolic fingerprint. More than 10 ions were detected and mass assignments were in agreement with predicted isotopic distributions for the intact compounds and related fragments; ions occurred as multiply protonated species and with common salt adducts. The most prevalent ion was observed at 919.88 m/z, which represents the aglycone [prymagly+2H](2+) backbone structure common to both molecules. Expanded mass spectra for this and related ions were in excellent agreement (<0.5ppm) with empirically derived spectra based on elemental composition and naturally occurring isotopes. These investigations have confirmed the isolation of polyketide prymnesins from whole cells, which heretofore has not been reproduced since their original characterization. Moreover, this study represents the first time these compounds have been verified in aqueous materials. These tools should allow the direct identification and analysis of polyketide prymnesins, which will greatly improve our understanding of these toxins in P. parvum. Copyright © 2013 Elsevier Inc. All rights reserved.

Condensation of Macrocyclic Polyketides Produced by Penicillium sp. DRF2 with Mercaptopyruvate Represents a New Fungal Detoxification Pathway.

PubMed

de Castro, Marcos V; Ióca, Laura P; Williams, David E; Costa, Bruna Z; Mizuno, Carolina M; Santos, Mario F C; de Jesus, Karen; Ferreira, Éverton L F; Seleghim, Mirna H R; Sette, Lara D; Pereira Filho, Edenir R; Ferreira, Antonio G; Gonçalves, Natália S; Santos, Raquel A; Patrick, Brian O; Andersen, Raymond J; Berlinck, Roberto G S

2016-06-24

Application of a refined procedure of experimental design and chemometric analysis to improve the production of curvularin-related polyketides by a marine-derived Penicillium sp. DRF2 resulted in the isolation and identification of cyclothiocurvularins 6-8 and cyclosulfoxicurvularins 10 and 11, novel curvularins condensed with a mercaptolactate residue. Two additional new curvularins, 3 and 4, are also reported. The structures of the sulfur-bearing curvularins were unambiguously established by analysis of spectroscopic data and by X-ray diffraction analysis. Analysis of stable isotope feeding experiments with [U-(13)C3(15)N]-l-cysteine confirmed the presence of the 2-hydroxy-3-mercaptopropanoic acid residue in 6-8 and the oxidized sulfoxide in 10 and 11. Cyclothiocurvularins A (6) and B (7) are formed by spontaneous reaction between 10,11-dehydrocurvularin (2) and mercaptopyruvate (12) obtained by transamination of cysteine. High ratios of [U-(13)C3(15)N]-l-cysteine incorporation into cyclothiocurvularin B (7), the isolation of two diastereomers of cyclothiocurvularins, the lack of cytotoxicity of cyclothiocurvularin B (7) and its methyl ester (8), and the spontaneous formation of cyclothiocurvularins from 10,11-dehydrocurvularin and mercaptopyruvate provide evidence that the formation of cyclothiocurvularins may well correspond to a 10,11-dehydrocurvularin detoxification process by Penicillium sp. DRF2.

Diversity of nonribosomal peptide synthetase and polyketide synthase gene clusters among taxonomically close Streptomyces strains.

PubMed

Komaki, Hisayuki; Sakurai, Kenta; Hosoyama, Akira; Kimura, Akane; Igarashi, Yasuhiro; Tamura, Tomohiko

2018-05-02

To identify the species of butyrolactol-producing Streptomyces strain TP-A0882, whole genome-sequencing of three type strains in a close taxonomic relationship was performed. In silico DNA-DNA hybridization using the genome sequences suggested that Streptomyces sp. TP-A0882 is classified as Streptomyces diastaticus subsp. ardesiacus. Strain TP-A0882, S. diastaticus subsp. ardesiacus NBRC 15402 T , Streptomyces coelicoflavus NBRC 15399 T , and Streptomyces rubrogriseus NBRC 15455 T harbor at least 14, 14, 10, and 12 biosynthetic gene clusters (BGCs), respectively, coding for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). All 14 gene clusters were shared by S. diastaticus subsp. ardesiacus strains TP-A0882 and NBRC 15402 T , while only four gene clusters were shared by the three distinct species. Although BGCs for bacteriocin, ectoine, indole, melanine, siderophores such as deferrioxamine, terpenes such as albaflavenone, hopene, carotenoid and geosmin are shared by the three species, many BGCs for secondary metabolites such as butyrolactone, lantipeptides, oligosaccharide, some terpenes are species-specific. These results indicate the possibility that strains belonging to the same species possess the same set of secondary metabolite-biosynthetic pathways, whereas strains belonging to distinct species have species-specific pathways, in addition to some common pathways, even if the strains are taxonomically close.

Polyketides with Immunosuppressive Activities from Mangrove Endophytic Fungus Penicillium sp. ZJ-SY₂.

PubMed

Liu, Hongju; Chen, Senhua; Liu, Weiyang; Liu, Yayue; Huang, Xishan; She, Zhigang

2016-11-25

Nine polyketides, including two new benzophenone derivatives, peniphenone ( 1 ) and methyl peniphenone ( 2 ), along with seven known xanthones ( 3 - 9 ) were obtained from mangrove endophytic fungus Penicillium sp. ZJ-SY₂ isolated from the leaves of Sonneratia apetala . Their structures were elucidated on the basis of MS, 1D, and 2D NMR data. Compounds 1 , 3 , 5 , and 7 showed potent immunosuppressive activity with IC 50 values ranging from 5.9 to 9.3 μg/mL.

Isolation and Assessment of the in Vitro Anti-Tumor Activity of Smenothiazole A and B, Chlorinated Thiazole-Containing Peptide/Polyketides from the Caribbean Sponge, Smenospongia aurea

PubMed Central

Esposito, Germana; Teta, Roberta; Miceli, Roberta; Ceccarelli, Luca S.; Della Sala, Gerardo; Camerlingo, Rosa; Irollo, Elena; Mangoni, Alfonso; Pirozzi, Giuseppe; Costantino, Valeria

2015-01-01

The study of the secondary metabolites contained in the organic extract of Caribbean sponge Smenospongia aurea led to the isolation of smenothiazole A (3) and B (4), hybrid peptide/polyketide compounds. Assays performed using four solid tumor cell lines showed that smenothiazoles exert a potent cytotoxic activity at nanomolar levels, with selectivity over ovarian cancer cells and a pro-apoptotic mechanism. PMID:25603342

Ruthenium-catalyzed insertion of adjacent diol carbon atoms into C-C bonds: Entry to type II polyketides.

PubMed

Bender, Matthias; Turnbull, Ben W H; Ambler, Brett R; Krische, Michael J

2017-08-25

Current catalytic processes involving carbon-carbon bond activation rely on π-unsaturated coupling partners. Exploiting the concept of transfer hydrogenative coupling, we report a ruthenium(0)-catalyzed cycloaddition of benzocyclobutenones that functionalizes two adjacent saturated diol carbon-hydrogen bonds. These regio- and diastereoselective processes enable convergent construction of type II polyketide substructures. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Biosynthesis of oxygen and nitrogen-containing heterocycles in polyketides

PubMed Central

Hemmerling, Franziska

2016-01-01

Summary This review highlights the biosynthesis of heterocycles in polyketide natural products with a focus on oxygen and nitrogen-containing heterocycles with ring sizes between 3 and 6 atoms. Heterocycles are abundant structural elements of natural products from all classes and they often contribute significantly to their biological activity. Progress in recent years has led to a much better understanding of their biosynthesis. In this context, plenty of novel enzymology has been discovered, suggesting that these pathways are an attractive target for future studies. PMID:27559404

On the generation of novel anticancer drugs by recombinant DNA technology: the use of combinatorial biosynthesis to produce novel drugs.

PubMed

Méndez, Carmen; Salas, José A

2003-09-01

Chemotherapeutic drugs for cancer treatment have been traditionally originated by the isolation of natural products from different environmental niches, by chemical synthesis or by a combination of both approaches thus generating semisynthetic drugs. In the last years, a number of gene clusters from several antitumor biosynthetic pathways, mainly produced by actinomycetes and belonging to the polyketides family, are being characterized. Genetic manipulation of these antitumor biosynthetic pathways will offer in the near future an alternative for the generation of novel antitumor derivatives and thus complementing current methods for obtaining novel anticancer drugs. Novel antitumor derivatives have been produced by targetted gene disruption and heterologous expression of single (or a few) gene(s) in another hosts or by combining genes from different, but structurally related, biosynthetic pathways ("combinatorial biosynthesis"). These strategies take advantage from the "relaxed substrate specificity" that characterize secondary metabolism enzymes.

Lasiodiplodia sp. ME4-2, an endophytic fungus from the floral parts of Viscum coloratum, produces indole-3-carboxylic acid and other aromatic metabolites.

PubMed

Qian, Chao-Dong; Fu, Yu-Hang; Jiang, Fu-Sheng; Xu, Zheng-Hong; Cheng, Dong-Qing; Ding, Bin; Gao, Cheng-Xian; Ding, Zhi-Shan

2014-11-30

Studies on endophytes, a relatively under-explored group of microorganisms, are currently popular amongst biologists and natural product researchers. A fungal strain (ME4-2) was isolated from flower samples of mistletoe (Viscum coloratum) during a screening program for endophytes. As limited information on floral endophytes is available, the aim of the present study is to characterise fungal endophytes using their secondary metabolites. ME4-2 grew well in both natural and basic synthetic media but produced no conidia. Sequence analysis of its internal transcribed spacer rDNA demonstrated that ME4-2 forms a distinct branch within the genus Lasiodiplodia and is closely related to L. pseudotheobromae. This floral endophyte was thus identified as Lasiodiplodia sp. based on its molecular biological characteristics. Five aromatic compounds, including cyclo-(Trp-Ala), indole-3-carboxylic acid (ICA), indole-3-carbaldehyde, mellein and 2-phenylethanol, were found in the culture. The structures of these compounds were determined using spectroscopic methods combined with gas chromatography. To the best of our knowledge, our work is the first to report isolation of these aromatic metabolites from a floral endophyte. Interestingly, ICA, a major secondary metabolite produced by ME4-2, seemed to be biosynthesized via an unusual pathway. Furthermore, our results indicate that the fungus ME4-2 is a potent producer of 2-phenylethanol, which is a common component of floral essential oils. This study introduces a fungal strain producing several important aromatic metabolites with pharmaceutical or food applications and suggests that endophytic fungi isolated from plant flowers are promising natural sources of aromatic compounds.

The Polyketide Synthase Gene pks4 of Trichoderma reesei Provides Pigmentation and Stress Resistance

PubMed Central

Atanasova, Lea; Knox, Benjamin P.; Kubicek, Christian P.; Baker, Scott E.

2013-01-01

Species of the fungal genus Trichoderma (Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichoderma polyketide synthase (PKS)-encoding genes, the pks4 from T. reesei was shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusarium spp. In this study, we show that deletion of this gene in T. reesei results in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reesei against other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4 significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi. PMID:24036343

Polyketide synthases from poison hemlock (Conium maculatum L.).

PubMed

Hotti, Hannu; Seppänen-Laakso, Tuulikki; Arvas, Mikko; Teeri, Teemu H; Rischer, Heiko

2015-11-01

Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by the condensation of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). We isolated PKS genes or their fragments from poison hemlock (Conium maculatum L.) by using random amplification of cDNA ends (RACE) and transcriptome analysis, and characterized three full-length enzymes by feeding different starter-CoAs in vitro. On the basis of our in vitro experiments, two of the three characterized PKS genes in poison hemlock encode chalcone synthases (CPKS1 and CPKS2), and one encodes a novel type of PKS (CPKS5). We show that CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. Our results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs. © 2015 FEBS.

Cyanobactins from Cyanobacteria: Current Genetic and Chemical State of Knowledge.

PubMed

Martins, Joana; Vasconcelos, Vitor

2015-11-13

Cyanobacteria are considered to be one of the most promising sources of new, natural products. Apart from non-ribosomal peptides and polyketides, ribosomally synthesized and post-translationally modified peptides (RiPPs) are one of the leading groups of bioactive compounds produced by cyanobacteria. Among these, cyanobactins have sparked attention due to their interesting bioactivities and for their potential to be prospective candidates in the development of drugs. It is assumed that the primary source of cyanobactins is cyanobacteria, although these compounds have also been isolated from marine animals such as ascidians, sponges and mollusks. The aim of this review is to update the current knowledge of cyanobactins, recognized as being produced by cyanobacteria, and to emphasize their genetic clusters and chemical structures as well as their bioactivities, ecological roles and biotechnological potential.

Process for detoxifying coal tars

DOEpatents

Longwell, John P.; Peters, William A.

1983-01-01

A process for treating liquid hydrocarbons to remove toxic, mutagenic and/or carcinogenic aromatic hydrocarbons comprises feeding the hydrocarbons into a reactor where vapors are thermally treated in contact with a catalyst consisting essentially of calcium oxide or a calcium oxide containing mineral. Thermally treating liquid hydrocarbons in contact with calcium oxide preferentially increases the cracking of aromatics thus producing a product having a reduced amount of aromatic compounds.

Effect of cerulenin on fatty acid composition and gene expression pattern of DHA-producing strain Colwellia psychrerythraea strain 34H.

PubMed

Wan, Xia; Peng, Yun-Feng; Zhou, Xue-Rong; Gong, Yang-Min; Huang, Feng-Hong; Moncalián, Gabriel

2016-02-06

Colwellia psychrerythraea 34H is a psychrophilic bacterium able to produce docosahexaenoic acid (DHA). Polyketide synthase pathway is assumed to be responsible for DHA production in marine bacteria. Five pfa genes from strain 34H were confirmed to be responsible for DHA formation by heterogeneous expression in Escherichia coli. The complexity of fatty acid profile of this strain was revealed by GC and GC-MS. Treatment of cells with cerulenin resulted in significantly reduced level of C16 monounsaturated fatty acid (C16:1(Δ9t), C16:1(Δ7)). In contrast, the amount of saturated fatty acids (C10:0, C12:0, C14:0), hydroxyl fatty acids (3-OH C10:0 and 3-OH C12:0), as well as C20:4ω3, C20:5ω3 and C22:6ω3 were increased. RNA sequencing (RNA-Seq) revealed the altered gene expression pattern when C. psychrerythraea cells were treated with cerulenin. Genes involved in polyketide synthase pathway and fatty acid biosynthesis pathway were not obviously affected by cerulenin treatment. In contrast, several genes involved in fatty acid degradation or β-oxidation pathway were dramatically reduced at the transcriptional level. Genes responsible for DHA formation in C. psychrerythraea was first cloned and characterized. We revealed the complexity of fatty acid profile in this DHA-producing strain. Cerulenin could substantially change the fatty acid composition by affecting the fatty acid degradation at transcriptional level. Acyl-CoA dehydrogenase gene family involved in the first step of β-oxidation pathway may be important to the selectivity of degraded fatty acids. In addition, inhibition of FabB protein by cerulenin may lead to the accumulation of malonyl-CoA, which is the substrate for DHA formation.

Saccharomyces cerevisiae Mixed Culture of Blackberry (Rubus ulmifolius L.) Juice: Synergism in the Aroma Compounds Production

PubMed Central

Ragazzo-Sánchez, Juan Arturo; Ortiz-Basurto, Rosa Isela; Luna-Solano, Guadalupe; Calderón-Santoyo, Montserrat

2014-01-01

Blackberry (Rubus sp.) juice was fermented using four different strains of Saccharomyces cerevisiae (Vitilevure-CM4457, Enoferm-T306, ICV-K1, and Greroche Rhona-L3574) recognized because of their use in the wine industry. A medium alcoholic graduation spirit (<6°GL) with potential to be produced at an industrial scale was obtained. Alcoholic fermentations were performed at 28°C, 200 rpm, and noncontrolled pH. The synergistic effect on the aromatic compounds production during fermentation in mixed culture was compared with those obtained by monoculture and physic mixture of spirits produced in monoculture. The aromatic composition was determined by HS-SPME-GC. The differences in aromatic profile principally rely on the proportions in aromatic compounds and not on the number of those compounds. The multivariance analysis, principal component analysis (PCA), and factorial discriminant analysis (DFA) permit to demonstrate the synergism between the strains. PMID:25506606

Pseudomonas syringae Phytotoxins: Mode of Action, Regulation, and Biosynthesis by Peptide and Polyketide Synthetases

PubMed Central

Bender, Carol L.; Alarcón-Chaidez, Francisco; Gross, Dennis C.

1999-01-01

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents. PMID:10357851

Anatomy of the β-branching enzyme of polyketide biosynthesis and its interaction with an acyl-ACP substrate.

PubMed

Maloney, Finn P; Gerwick, Lena; Gerwick, William H; Sherman, David H; Smith, Janet L

2016-09-13

Alkyl branching at the β position of a polyketide intermediate is an important variation on canonical polyketide natural product biosynthesis. The branching enzyme, 3-hydroxy-3-methylglutaryl synthase (HMGS), catalyzes the aldol addition of an acyl donor to a β-keto-polyketide intermediate acceptor. HMGS is highly selective for two specialized acyl carrier proteins (ACPs) that deliver the donor and acceptor substrates. The HMGS from the curacin A biosynthetic pathway (CurD) was examined to establish the basis for ACP selectivity. The donor ACP (CurB) had high affinity for the enzyme (Kd = 0.5 μM) and could not be substituted by the acceptor ACP. High-resolution crystal structures of HMGS alone and in complex with its donor ACP reveal a tight interaction that depends on exquisite surface shape and charge complementarity between the proteins. Selectivity is explained by HMGS binding to an unusual surface cleft on the donor ACP, in a manner that would exclude the acceptor ACP. Within the active site, HMGS discriminates between pre- and postreaction states of the donor ACP. The free phosphopantetheine (Ppant) cofactor of ACP occupies a conserved pocket that excludes the acetyl-Ppant substrate. In comparison with HMG-CoA (CoA) synthase, the homologous enzyme from primary metabolism, HMGS has several differences at the active site entrance, including a flexible-loop insertion, which may account for the specificity of one enzyme for substrates delivered by ACP and the other by CoA.

Recognition of Acyl Carrier Proteins by Ketoreductases in Assembly Line Polyketide Synthases

PubMed Central

Ostrowski, Matthew P.; Cane, David E.; Khosla, Chaitan

2016-01-01

Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed a ca. 10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed a ca. 10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules. PMID:27118242

iso-Migrastatin, migrastatin, and dorrigocin production in Streptomyces platensis NRRL 18993 is governed by a single biosynthetic machinery featuring an acyltransferase-less type I polyketide synthase.

PubMed

Lim, Si-Kyu; Ju, Jianhua; Zazopoulos, Emmanuel; Jiang, Hui; Seo, Jeong-Woo; Chen, Yihua; Feng, Zhiyang; Rajski, Scott R; Farnet, Chris M; Shen, Ben

2009-10-23

iso-Migrastatin and related glutarimide-containing polyketides are potent inhibitors of tumor cell migration and their implied potential as antimetastatic agents for human cancers has garnered significant attention. Genome scanning of Streptomyces platensis NRRL 18993 unveiled two candidate gene clusters (088D and mgs); each encodes acyltransferase-less type I polyketide synthases commensurate with iso-migrastatin biosynthesis. Both clusters were inactivated by lambda-RED-mediated PCR-targeting mutagenesis in S. platensis; iso-migrastatin production was completely abolished in the DeltamgsF mutant SB11012 strain, whereas inactivation of 088D-orf7 yielded the SB11006 strain that exhibited no discernible change in iso-migrastatin biosynthesis. These data indicate that iso-migrastatin production is governed by the mgs cluster. Systematic gene inactivation allowed determination of the precise boundaries of the mgs cluster and the essentiality of the genes within the mgs cluster in iso-migrastatin production. The mgs cluster consists of 11 open reading frames that encode three acyltransferase-less type I polyketide synthases (MgsEFG), one discrete acyltransferase (MgsH), a type II thioesterase (MgsB), three post-PKS tailoring enzymes (MgsIJK), two glutarimide biosynthesis enzymes (MgsCD), and one regulatory protein (MgsA). A model for iso-migrastatin biosynthesis is proposed based on functional assignments derived from bioinformatics and is further supported by the results of in vivo gene inactivation experiments.

iso-Migrastatin, Migrastatin, and Dorrigocin Production in Streptomyces platensis NRRL 18993 Is Governed by a Single Biosynthetic Machinery Featuring an Acyltransferase-less Type I Polyketide Synthase*

PubMed Central

Lim, Si-Kyu; Ju, Jianhua; Zazopoulos, Emmanuel; Jiang, Hui; Seo, Jeong-Woo; Chen, Yihua; Feng, Zhiyang; Rajski, Scott R.; Farnet, Chris M.; Shen, Ben

2009-01-01

iso-Migrastatin and related glutarimide-containing polyketides are potent inhibitors of tumor cell migration and their implied potential as antimetastatic agents for human cancers has garnered significant attention. Genome scanning of Streptomyces platensis NRRL 18993 unveiled two candidate gene clusters (088D and mgs); each encodes acyltransferase-less type I polyketide synthases commensurate with iso-migrastatin biosynthesis. Both clusters were inactivated by λ-RED-mediated PCR-targeting mutagenesis in S. platensis; iso-migrastatin production was completely abolished in the ΔmgsF mutant SB11012 strain, whereas inactivation of 088D-orf7 yielded the SB11006 strain that exhibited no discernible change in iso-migrastatin biosynthesis. These data indicate that iso-migrastatin production is governed by the mgs cluster. Systematic gene inactivation allowed determination of the precise boundaries of the mgs cluster and the essentiality of the genes within the mgs cluster in iso-migrastatin production. The mgs cluster consists of 11 open reading frames that encode three acyltransferase-less type I polyketide synthases (MgsEFG), one discrete acyltransferase (MgsH), a type II thioesterase (MgsB), three post-PKS tailoring enzymes (MgsIJK), two glutarimide biosynthesis enzymes (MgsCD), and one regulatory protein (MgsA). A model for iso-migrastatin biosynthesis is proposed based on functional assignments derived from bioinformatics and is further supported by the results of in vivo gene inactivation experiments. PMID:19726666

Further insights into brevetoxin metabolism by de novo radiolabeling.

PubMed

Calabro, Kevin; Guigonis, Jean-Marie; Teyssié, Jean-Louis; Oberhänsli, François; Goudour, Jean-Pierre; Warnau, Michel; Bottein, Marie-Yasmine Dechraoui; Thomas, Olivier P

2014-06-10

The toxic dinoflagellate Karenia brevis, responsible for early harmful algal blooms in the Gulf of Mexico, produces many secondary metabolites, including potent neurotoxins called brevetoxins (PbTx). These compounds have been identified as toxic agents for humans, and they are also responsible for the deaths of several marine organisms. The overall biosynthesis of these highly complex metabolites has not been fully ascertained, even if there is little doubt on a polyketide origin. In addition to gaining some insights into the metabolic events involved in the biosynthesis of these compounds, feeding studies with labeled precursors helps to discriminate between the de novo biosynthesis of toxins and conversion of stored intermediates into final toxic products in the response to environmental stresses. In this context, the use of radiolabeled precursors is well suited as it allows working with the highest sensitive techniques and consequently with a minor amount of cultured dinoflagellates. We were then able to incorporate [U-¹⁴C]-acetate, the renowned precursor of the polyketide pathway, in several PbTx produced by K. brevis. The specific activities of PbTx-1, -2, -3, and -7, identified by High-Resolution Electrospray Ionization Mass Spectrometer (HRESIMS), were assessed by HPLC-UV and highly sensitive Radio-TLC counting. We demonstrated that working at close to natural concentrations of acetate is a requirement for biosynthetic studies, highlighting the importance of highly sensitive radiolabeling feeding experiments. Quantification of the specific activity of the four, targeted toxins led us to propose that PbTx-1 and PbTx-2 aldehydes originate from oxidation of the primary alcohols of PbTx-7 and PbTx-3, respectively. This approach will open the way for a better comprehension of the metabolic pathways leading to PbTx but also to a better understanding of their regulation by environmental factors.

Further Insights into Brevetoxin Metabolism by de Novo Radiolabeling

PubMed Central

Calabro, Kevin; Guigonis, Jean-Marie; Teyssié, Jean-Louis; Oberhänsli, François; Goudour, Jean-Pierre; Warnau, Michel; Dechraoui Bottein, Marie-Yasmine; Thomas, Olivier P.

2014-01-01

The toxic dinoflagellate Karenia brevis, responsible for early harmful algal blooms in the Gulf of Mexico, produces many secondary metabolites, including potent neurotoxins called brevetoxins (PbTx). These compounds have been identified as toxic agents for humans, and they are also responsible for the deaths of several marine organisms. The overall biosynthesis of these highly complex metabolites has not been fully ascertained, even if there is little doubt on a polyketide origin. In addition to gaining some insights into the metabolic events involved in the biosynthesis of these compounds, feeding studies with labeled precursors helps to discriminate between the de novo biosynthesis of toxins and conversion of stored intermediates into final toxic products in the response to environmental stresses. In this context, the use of radiolabeled precursors is well suited as it allows working with the highest sensitive techniques and consequently with a minor amount of cultured dinoflagellates. We were then able to incorporate [U-14C]-acetate, the renowned precursor of the polyketide pathway, in several PbTx produced by K. brevis. The specific activities of PbTx-1, -2, -3, and -7, identified by High-Resolution Electrospray Ionization Mass Spectrometer (HRESIMS), were assessed by HPLC-UV and highly sensitive Radio-TLC counting. We demonstrated that working at close to natural concentrations of acetate is a requirement for biosynthetic studies, highlighting the importance of highly sensitive radiolabeling feeding experiments. Quantification of the specific activity of the four, targeted toxins led us to propose that PbTx-1 and PbTx-2 aldehydes originate from oxidation of the primary alcohols of PbTx-7 and PbTx-3, respectively. This approach will open the way for a better comprehension of the metabolic pathways leading to PbTx but also to a better understanding of their regulation by environmental factors. PMID:24918358

Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea.

PubMed

Cortés, Jesús; Velasco, Javier; Foster, Graham; Blackaby, Andrew P; Rudd, Brian A M; Wilkinson, Barrie

2002-06-01

The soluble, diffusible red-brown pigment produced by a Saccharopolyspora erythraea "red variant" has been shown to contain glycosylated and polymerized derivatives of 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). Flaviolin is a spontaneous oxidation product of 1,3,6,8-tetrahydroxynaphthalene (THN), which is biosynthesized in bacteria by a chalcone synthase-like (CS-like) type III polyketide synthase (PKS). A fragment of the gene responsible for THN biosynthesis in S. erythraea E_8-7 was amplified by polymerase chain reaction (PCR) using degenerate primers based on conserved regions of known plant CS and bacterial CS-like genes. From the isolated fragment, a suicide vector was prepared, which was subsequently used to disrupt the red-brown pigment-producing (rpp) locus in S. erythraea, generating a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant was subsequently used in a vector-recapture protocol to isolate a plasmid that contained an insert spanning the entire rpp locus. Sequencing of the insert revealed that the disrupted open reading frame (ORF) encodes a CS-like protein displaying 69% sequence identity to the rppA gene of Streptomyces griseus. The S. griseus rppA gene encodes RppA, the first characterized bacterial CS-like protein, which is sufficient in vitro for the synthesis of THN from malonyl-CoA. The rppA disruption mutant and rppA sequence provided a means by which to address the mechanism of diffusible pigment biosynthesis, as well as to investigate any link between this and the modulation of erythromycin A titre, which has been observed for S. erythraea variants.

Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa

PubMed Central

Morita, Hiroyuki; Wanibuchi, Kiyofumi; Nii, Hirohiko; Kato, Ryohei; Sugio, Shigetoshi; Abe, Ikuro

2010-01-01

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C6-C7-C6 diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C6-C3 coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H2O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C6-C7-C6 scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes. PMID:21041675

Diverse and Abundant Secondary Metabolism Biosynthetic Gene Clusters in the Genomes of Marine Sponge Derived Streptomyces spp. Isolates.

PubMed

Jackson, Stephen A; Crossman, Lisa; Almeida, Eduardo L; Margassery, Lekha Menon; Kennedy, Jonathan; Dobson, Alan D W

2018-02-20

The genus Streptomyces produces secondary metabolic compounds that are rich in biological activity. Many of these compounds are genetically encoded by large secondary metabolism biosynthetic gene clusters (smBGCs) such as polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) which are modular and can be highly repetitive. Due to the repeats, these gene clusters can be difficult to resolve using short read next generation datasets and are often quite poorly predicted using standard approaches. We have sequenced the genomes of 13 Streptomyces spp. strains isolated from shallow water and deep-sea sponges that display antimicrobial activities against a number of clinically relevant bacterial and yeast species. Draft genomes have been assembled and smBGCs have been identified using the antiSMASH (antibiotics and Secondary Metabolite Analysis Shell) web platform. We have compared the smBGCs amongst strains in the search for novel sequences conferring the potential to produce novel bioactive secondary metabolites. The strains in this study recruit to four distinct clades within the genus Streptomyces . The marine strains host abundant smBGCs which encode polyketides, NRPS, siderophores, bacteriocins and lantipeptides. The deep-sea strains appear to be enriched with gene clusters encoding NRPS. Marine adaptations are evident in the sponge-derived strains which are enriched for genes involved in the biosynthesis and transport of compatible solutes and for heat-shock proteins. Streptomyces spp. from marine environments are a promising source of novel bioactive secondary metabolites as the abundance and diversity of smBGCs show high degrees of novelty. Sponge derived Streptomyces spp. isolates appear to display genomic adaptations to marine living when compared to terrestrial strains.

Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa.

PubMed

Morita, Hiroyuki; Wanibuchi, Kiyofumi; Nii, Hirohiko; Kato, Ryohei; Sugio, Shigetoshi; Abe, Ikuro

2010-11-16

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C(6)-C(7)-C(6) diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C(6)-C(3) coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H(2)O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C(6)-C(7)-C(6) scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes.

Cloning and Characterization of the Tetrocarcin A Gene Cluster from Micromonospora chalcea NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics▿ †

PubMed Central

Fang, Jie; Zhang, Yiping; Huang, Lijuan; Jia, Xinying; Zhang, Qi; Zhang, Xu; Tang, Gongli; Liu, Wen

2008-01-01

Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of d-tetronitrose, l-amicetose, and l-digitoxose may begin with d-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to ΔchlD3 and ΔchlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity. PMID:18586939

Biosurfactant produced by novel Pseudomonas sp. WJ6 with biodegradation of n-alkanes and polycyclic aromatic hydrocarbons.

PubMed

Xia, Wenjie; Du, Zhifeng; Cui, Qingfeng; Dong, Hao; Wang, Fuyi; He, Panqing; Tang, YongChun

2014-07-15

Alkanes and polycyclic aromatic hydrocarbons (PAHs) have threatened the environment due to toxicity and poor bioavailability. Interest in degradation of these hazardous materials by biosurfactant-producing bacteria has been steadily increasing in recent years. In this work, a novel biosurfactant-producing Pseudomonas sp. WJ6 was isolated to degrade a wide range of n-alkanes and polycyclic aromatic hydrocarbons. Production of lipopeptide biosurfactant was observed in all biodegradable studies. These lipopeptides were purified and identified by C18 RP-HPLC system and electrospray ionization-mass spectrometry. Results of structural analysis showed that these lipopeptides generated from different hydrocarbons were classified to be surfactin, fengycin and lichenysin. Heavy-oil sludge washing experiments demonstrated that lipopeptides produced by Pseudomonas sp. WJ6 have 92.46% of heavy-oil washing efficiency. The obtained results indicate that this novel bacterial strain and its lipopeptides have great potentials in the environmental remediation and petroleum recovery. Copyright © 2014 Elsevier B.V. All rights reserved.

Automated analysis of oxidative metabolites

NASA Technical Reports Server (NTRS)

Furner, R. L. (Inventor)

1974-01-01

An automated system for the study of drug metabolism is described. The system monitors the oxidative metabolites of aromatic amines and of compounds which produce formaldehyde on oxidative dealkylation. It includes color developing compositions suitable for detecting hyroxylated aromatic amines and formaldehyde.

Chemical recalcitrance of biochar and wildfire charcoal: how similar are they?

NASA Astrophysics Data System (ADS)

Santin, Cristina; Doerr, Stefan H.; Merino, Agustin

2016-04-01

The enhanced chemical resistance to biological degradation of pyrogenic materials, either produced during wildfires (charcoal) or by man (biochar), makes them long-term carbon sinks once incorporated in soils. In spite of their fundamental similarities, studies comparing the chemical recalcitrance of biochar and wildfire charcoal are scarce because analogous materials for accurate comparison are not easily available. Using solid-state 13C cross polarization-magic angle spinning nuclear magnetic resonance spectroscopy we characterized the chemical recalcitrance of pyrogenic materials generated from the same unburnt feedstooks (litter and dead wood from Pinus banksiana): (a) charcoal from a high-intensity wildfire and (b) biochar obtained by slow pyrolysis [3 treatments: 2 h at 350, 500 and 650°C]. For quantification, the spectra were divided into four regions representing different chemical environments of the 13C nucleus: alkyl C (0-45 ppm), O-alkyl C (45-110 ppm), olefinic and aromatic C(110-160 ppm), and carbonyl C (160-210 ppm). As an indicator of chemical recalcitrance, the degree of aromaticity (%) was calculated as follow: aromatic-C ∗ 100 / (alkyl C+ O alkyl-C + aromatic-C). The pyrogenic materials derived from wood show higher degrees of aromaticity (68 to 88%) than pyrogenic material derived from litter (40 to 88%). When comparing biochar and wildfire charcoal, biochars produced at 500 and 650°C always have higher degrees of aromaticity than wildfire charcoals, irrespective of the original feedstock. Wildfire charcoals always show a more heterogeneous chemical composition, with alkyl and O-alkyl compounds present even in charcoal generated at very high temperatures (temperatures up to 950 °C were recorded on the litter surface during the wildfire). However, biochars produced at 500 and 650 °C are mostly aromatic, and only the biochars produced at 350 °C show partial contribution of alkyl-C compounds. Our results suggest that biochar-type pyrogenic materials have in general a higher chemical recalcitrance than wildfire charcoal and, thus, we advice caution when transfer knowledge between the biochar and the wildfire charcoal research communities.

The production of aromatic alcohols in non-Saccharomyces wine yeast is modulated by nutrient availability.

PubMed

González, Beatriz; Vázquez, Jennifer; Morcillo-Parra, M Ángeles; Mas, Albert; Torija, María Jesús; Beltran, Gemma

2018-09-01

Aromatic alcohols (tryptophol, phenylethanol, tyrosol) positively contribute to organoleptic characteristics of wines, and are also described as bioactive compounds and quorum sensing molecules. These alcohols are produced by yeast during alcoholic fermentation via the Erhlich pathway, although in non-Saccharomyces this production has been poorly studied. We studied how different wine yeast species modulate the synthesis patterns of aromatic alcohol production depending on glucose, nitrogen and aromatic amino acid availability. Nitrogen limitation strongly promoted the production of aromatic alcohols in all strains, whereas low glucose generally inhibited it. Increased aromatic amino acid concentrations stimulated the production of aromatic alcohols in all of the strains and conditions tested. Thus, there was a clear association between the nutrient conditions and production of aromatic alcohols in most of the wine yeast species analysed. Additionally, the synthesis pattern of these alcohols has been evaluated for the first time in Torulaspora delbrueckii, Metschnikowia pulcherrima and Starmellera bacillaris. Copyright © 2018 Elsevier Ltd. All rights reserved.

Polyimide Foams

NASA Technical Reports Server (NTRS)

Vazquez, Juan M. (Inventor); Cano, Roberto J. (Inventor); Jensen, Brian J. (Inventor); Weiser, Erik S. (Inventor)

2005-01-01

A fully imidized, solvent-free polyimide foam having excellent mechanical, acoustic, thermal, and flame resistant properties is produced. A first solution is provided, which includes one or more aromatic dianhydrides or derivatives of aromatic dianhydrides, and may include one or more aromatic diamines, dissolved in one or more polar solvents, along with an effective amount of one or more blowing agents. This first solution may also advantageously include effective amounts respectively of one or mores catalysts, one or more surfactants, and one or more fire retardants. A second solution is also provided which includes one or more isocyanates. The first and second solutions are rapidly and thoroughly mixed to produce an admixture, which is allowed to foam-in an open container, or in a closed mold-under ambient conditions to completion produce a foamed product. This foamed product is then cured by high frequency electromagnetic radiation, thermal energy, or a combination thereof. Alternatively, the process is adapted for spraying or extrusion.

Polyimide foams

NASA Technical Reports Server (NTRS)

Weiser, Erik S. (Inventor); Cano, Roberto J. (Inventor); Jensen, Brian J. (Inventor); Vazquez, Juan M. (Inventor)

2005-01-01

A fully imidized, solvent-free polyimide foam having excellent mechanical, acoustic, thermal, and flame resistant properties is produced. A first solution is provided, which includes one or more aromatic dianhydrides or derivatives of aromatic dianhydrides, and may include one or more aromatic diamines, dissolved in one or more polar solvents, along with an effective amount of one or more blowing agents. This first solution may also advantageously include effective amounts respectively of one or mores catalysts, one or more surfactants, and one or more fire retardants. A second solution is also provided which includes one or more isocyanates. The first and second solutions are rapidly and thoroughly mixed to produce an admixture, which is allowed to foam?in an open container, or in a closed mold?under ambient conditions to completion produce a foamed product. This foamed product is then cured by high frequency electromagnetic radiation, thermal energy, or a combination thereof. Alternatively, the process is adapted for spraying or extrusion.

Polyimide foams

NASA Technical Reports Server (NTRS)

Vazquez, Juan M. (Inventor); Cano, Roberto J. (Inventor); Weiser, Erik S. (Inventor); Jensen, Brian J. (Inventor)

2009-01-01

A fully imidized, solvent-free polyimide foam having excellent mechanical, acoustic, thermal, and flame resistant properties is produced. A first solution is provided, which includes one or more aromatic dianhydrides or derivatives of aromatic dianhydrides, and may include one or more aromatic diamines, dissolved in one or more polar solvents, along with an effective amount of one or more blowing agents. This first solution may also advantageously include effective amounts respectively of one or mores catalysts, one or more surfactants, and one or more fire retardants. A second solution is also provided which includes one or more isocyanates. The first and second solutions are rapidly and thoroughly mixed to produce an admixture, which is allowed to foam--in an open container, or in a closed mold--under ambient conditions to completion produce a foamed product. This foamed product is then cured by high frequency electromagnetic radiation, thermal energy, or a combination thereof. Alternatively, the process is adapted for spraying or extrusion.

Penicyrones A and B, an epimeric pair of α-pyrone-type polyketides produced by the marine-derived Penicillium sp.

PubMed

Bu, Ying-Yue; Yamazaki, Hiroyuki; Takahashi, Ohgi; Kirikoshi, Ryota; Ukai, Kazuyo; Namikoshi, Michio

2016-01-01

Two polyketides containing an α-pyrone unit, named penicyrones A (1) and B (2), were isolated from a culture broth of the marine-derived Penicillium sp. TPU1271 together with nine known compounds: verrucosidin (3), fructigenine A (4), verrucofortine (5), cyclo-(L-Trp-L-Phe) (6), cyclopenol (7), cyclopenin (8), penipratynolene (9), aspterric acid (10) and viridicatol (11). The structures of 1 and 2 were elucidated by analyzing the spectroscopic data of 1, 2 and their O-acetyl derivatives (1a and 2a). Compounds 1 and 2 were epimers of each other at the C-9 position. The absolute configurations of 1 and 2 were assigned on the basis of NOESY data for 1, 2, 1a and 2a, a conformational analysis and the identity of the biogenetic pathway with verrucosidin (3). The planar structure of penicyrones was found in the SciFinder as a compound in the commercial chemical libraries; however, the stereostructure and spectroscopic data were not available. Therefore, this is the first study on the isolation and structure elucidation, including the absolute configurations, of penicyrones A (1) and B (2) as fungal metabolites. Compound 3 exhibited growth inhibitory activity against Mycobacterium smegmatis at 40 μg per disc (inhibition zone of 11 mm). This is the first study to demonstrate that verrucosidin (3) exhibited anti-mycobacterial activity.

Immunomodulatory Polyketides from a Phoma-like Fungus Isolated from a Soft Coral.

PubMed

Sun, Yi-Zhe; Kurtán, Tibor; Mándi, Attila; Tang, Hua; Chou, Yalan; Soong, Keryea; Su, Li; Sun, Peng; Zhuang, Chun-Lin; Zhang, Wen

2017-11-22

Fourteen new polyketides with a trans-fused decalin ring system, libertalides A-N (3-16), together with two known analogues, aspermytin A and its acetate (1, 2), were isolated from the fermentation extract of a coral-derived Libertasomyces sp. fungus. Their relative configurations were elucidated on the basis of detailed spectroscopic analysis, and the absolute configurations were determined by TDDFT-ECD and optical rotation (OR) calculations. The OR of 1 and 2 were found to have opposite signs in CH 3 CN and CHCl 3 , which was in agreement with the OR calculations producing alternating signs for the optical rotation depending on the applied conditions. Because the signs of the OR for 1 and 2 showed high solvent dependence, they may not be used alone to correlate the absolute configurations. Compound 16 displayed structural novelty characterized by an α-enol ether bridge conjugated with an aldehyde group. In in vitro immunomodulatory screening, compounds 1, 4, and 10 significantly induced the proliferation of CD3 + T cells, while compounds 2, 7, 11, and 14 significantly increased the CD4 + /CD8 + ratio at 3 μM. A preliminary structure-activity analysis revealed a crucial role of Δ 7 and a terminal OH group in the regulation of CD3 + T cell proliferation. This is the first report of immunoregulatory activity for metabolites of this kind.

Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome

PubMed Central

Müller, Christina A.; Oberauner-Wappis, Lisa; Peyman, Armin; Amos, Gregory C. A.; Wellington, Elizabeth M. H.

2015-01-01

Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications. PMID:26002894

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

PubMed Central

Jiang, Chunyan; Wang, Hougen; Kang, Qianjin; Liu, Jing

2012-01-01

Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity. PMID:22156425

Synthetic biology approaches to fluorinated polyketides

PubMed Central

Thuronyi, Benjamin W.; Chang, Michelle C. Y.

2016-01-01

Conspectus The catalytic diversity of living systems offers a broad range of opportunities for developing new methods to produce small molecule targets such as fuels, materials, and pharmaceuticals. In addition to providing cost-effective and renewable methods for large-scale commercial processes, the exploration of the unusual chemical phenotypes found in living organisms can also enable the expansion of chemical space for discovery of novel function by combining orthogonal attributes from both synthetic and biological chemistry. In this context, we have focused on the development of new fluorine chemistry using synthetic biology approaches. While fluorine has become an important feature in compounds of synthetic origin, the scope of biological fluorine chemistry in living systems is limited, with fewer than 20 organofluorine natural products identified to date. In order to expand the diversity of biosynthetically accessible organofluorines, we have begun to develop methods for the site-selective introduction of fluorine into complex natural products by engineering biosynthetic machinery to incorporate fluorinated building blocks. To gain insight into how both enzyme active sites and metabolic pathways can be evolved to manage and select for fluorinated compounds, we have studied one of the only characterized natural hosts for organofluorine biosynthesis, the soil microbe Streptomyces cattleya. This information provides a template for designing engineered organofluorine enzymes, pathways, and hosts and has allowed us to initiate construction of enzymatic and cellular pathways for the production of fluorinated polyketides. PMID:25719427

Direct Capture and Heterologous Expression of Salinispora Natural Product Genes for the Biosynthesis of Enterocin

PubMed Central

2015-01-01

Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora’s promising secondary metabolome. PMID:25382643

Direct capture and heterologous expression of Salinispora natural product genes for the biosynthesis of enterocin.

PubMed

Bonet, Bailey; Teufel, Robin; Crüsemann, Max; Ziemert, Nadine; Moore, Bradley S

2015-03-27

Heterologous expression of secondary metabolic pathways is a promising approach for the discovery and characterization of bioactive natural products. Herein we report the first heterologous expression of a natural product from the model marine actinomycete genus Salinispora. Using the recently developed method of yeast-mediated transformation-associated recombination for natural product gene clusters, we captured a type II polyketide synthase pathway from Salinispora pacifica with high homology to the enterocin pathway from Streptomyces maritimus and successfully produced enterocin in two different Streptomyces host strains. This result paves the way for the systematic interrogation of Salinispora's promising secondary metabolome.

An easy-to-perform photometric assay for methyltransferase activity measurements.

PubMed

Schäberle, Till F; Siba, Christian; Höver, Thomas; König, Gabriele M

2013-01-01

Methyltransferases (MTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a suitable substrate. Such methylations are important modifications in secondary metabolisms, especially on natural products produced by polyketide synthases and nonribosomal peptide synthetases, many of which are of special interest due to their prominent pharmacological activities (e.g., lovastatin, cyclosporin). To gain basic biochemical knowledge on the methylation process, it is of immense relevance to simplify methods concerning experimental problems caused by a large variety in substrates. Here, we present a photometric method to analyze MT activity by measuring SAM consumption in a coupled enzyme assay. Copyright © 2012 Elsevier Inc. All rights reserved.

Effects of Volatile Aromatic Anesthetics on Voltage-Gated Na+ Channels Expressed in Xenopus Oocytes

PubMed Central

Horishita, Takafumi; Eger, Edmond I; Harris, R. Adron

2008-01-01

Background Many inhaled anesthetics inhibit voltage-gated sodium channels at clinically relevant concentrations, and suppression of neurotransmitter release by these agents results, at least partly, from decreased presynaptic sodium channel activity. Volatile aromatic anesthetics can inhibit N-methyl-D-aspartate (NMDA) receptor function and enhance γ-amino butyric acid A (GABAA) receptor function, but these effects depend strongly on the chemical properties of the aromatic ompounds. The present study tested whether diverse aromatic anesthetics consistently inhibit sodium channel function. Methods We studied the effect of eight aromatic anesthetics on Nav1.2 sodium channels with β1 subunits, using whole-cell, two-electrode voltage-clamp techniques in Xenopus oocytes. Results All aromatic anesthetics inhibited INa (sodium currents) at a holding potential which produce half-maximal current (V1/2) (partial depolarization); inhibition was modest with 1,3,5-trifluorobenzene (8 ± 2%), pentafluorobenzene (13 ± 2%), and hexafluorobenzene (13 ± 2%), but greater with benzene (37 ± 2%), fluorobenzene (39 ± 2%), 1,2-difluorobenzene (48 ± 2%), 1,4-difluorobenzene (31 ± 3%), and 1,2,4-trifluorobenzene (33 ± 1%). Such dichotomous effects were noted by others for NMDA and GABAA receptors. Parallel, but much smaller inhibition, was found for INa at a holding potential which produced near maximal current (−90 mV) (VH-90), and hexafluorobenzene caused small (6 ± 1%) potentiation of this current. These changes in sodium channel function were correlated with effectiveness for inhibiting NMDA receptors, with lipid solubility of the compounds, with molecular volume, and with cation-π interactions. Conclusion Aromatic compounds vary in their actions on the kinetics of sodium channel gating and this may underlie their variable inhibition. The range of inhibition produced by MAC concentrations of inhaled anesthetics indicates that sodium channel inhibition may underlie the action of some of these anesthetics but not others. PMID:18931215

Cyanobactins from Cyanobacteria: Current Genetic and Chemical State of Knowledge

PubMed Central

Martins, Joana; Vasconcelos, Vitor

2015-01-01

Cyanobacteria are considered to be one of the most promising sources of new, natural products. Apart from non-ribosomal peptides and polyketides, ribosomally synthesized and post-translationally modified peptides (RiPPs) are one of the leading groups of bioactive compounds produced by cyanobacteria. Among these, cyanobactins have sparked attention due to their interesting bioactivities and for their potential to be prospective candidates in the development of drugs. It is assumed that the primary source of cyanobactins is cyanobacteria, although these compounds have also been isolated from marine animals such as ascidians, sponges and mollusks. The aim of this review is to update the current knowledge of cyanobactins, recognized as being produced by cyanobacteria, and to emphasize their genetic clusters and chemical structures as well as their bioactivities, ecological roles and biotechnological potential. PMID:26580631

A singular enzymatic megacomplex from Bacillus subtilis.

PubMed

Straight, Paul D; Fischbach, Michael A; Walsh, Christopher T; Rudner, David Z; Kolter, Roberto

2007-01-02

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

Informatic analysis reveals Legionella as a source of novel natural products.

PubMed

Johnston, Chad W; Plumb, Jonathan; Li, Xiang; Grinstein, Sergio; Magarvey, Nathan A

2016-06-01

Microbial natural products are a crucial source of bioactive molecules and unique chemical scaffolds. Despite their importance, rediscovery of known natural products from established productive microbes has led to declining interest, even while emergent genomic data suggest that the majority of microbial natural products remain to be discovered. Now, new sources of microbial natural products must be defined in order to provide chemical scaffolds for the next generation of small molecules for therapeutic, agricultural, and industrial purposes. In this work, we use specialized bioinformatic programs, genetic knockouts, and comparative metabolomics to define the genus Legionella as a new source of novel natural products. We show that Legionella spp. hold a diverse collection of biosynthetic gene clusters for the production of polyketide and nonribosomal peptide natural products. To confirm this bioinformatic survey, we create targeted mutants of L. pneumophila and use comparative metabolomics to identify a novel polyketide surfactant. Using spectroscopic techniques, we show that this polyketide possesses a new chemical scaffold, and firmly demonstrate that this unexplored genus is a source for novel natural products.

Improved heterologous production of the nonribosomal peptide-polyketide siderophore yersiniabactin through metabolic engineering and induction optimization.

PubMed

Ahmadi, Mahmoud Kamal; Pfeifer, Blaine A

2016-11-01

Biosynthesis of complex natural products like polyketides and nonribosomal peptides using Escherichia coli as a heterologous host provides an opportunity to access these molecules. The value in doing so stems from the fact that many compounds hold some therapeutic or other beneficial property and their original production hosts are intractable for a variety of reasons. In this work, metabolic engineering and induction variable optimization were used to increase production of the polyketide-nonribosomal peptide compound yersiniabactin, a siderophore that has been utilized to selectively remove metals from various solid and aqueous samples. Specifically, several precursor substrate support pathways were altered through gene expression and exogenous supplementation in order to boost production of the final compound. The gene expression induction process was also analyzed to identify the temperatures and inducer concentrations resulting in highest final production levels. When combined, yersiniabactin production was extended to ∼175 mg L -1 . © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1412-1417, 2016. © 2016 American Institute of Chemical Engineers.

Chaetochromones A and B, two new polyketides from the fungus Chaetomium indicum (CBS.860.68).

PubMed

Lu, Keyang; Zhang, Yisheng; Li, Li; Wang, Xuewei; Ding, Gang

2013-09-05

Chaetochromones A (1) and B (2), two novel polyketides, were isolated from the crude extract of fungus Chaetomium indicum (CBS.860.68) together with three known analogues PI-3(3), PI-4 (4) and SB236050 (5). The structures of these compounds were determined by HRESI-MS and NMR experiments. Chaetochromones A (1) and B (2) are a member of the polyketides family, which might originate from a similar biogenetic pathway as the known compounds PI-3 (3), PI-4 (4) and SB236050 (5). The biological activities of these secondary metabolites were evaluated against eight plant pathogens, including Alternaria alternata, Ilyonectria radicicola, Trichoderma viride pers, Aspergillus niger, Fusarium verticillioide, Irpex lacteus (Fr.), Poria placenta (Fr.) Cooke and Coriolus versicolor (L.) Quél. Compound 1 displayed moderate inhibitory rate (>60%) against the brown rot fungus Poria placenta (Fr.) Cooke, which causes significant wood decay. In addition, the cytotoxic activities against three cancer cell lines A549, MDA-MB-231, PANC-1 were also tested, without any inhibitory activities being detected.

Medicinally important aromatic plants with radioprotective activity

PubMed Central

Samarth, Ravindra M; Samarth, Meenakshi; Matsumoto, Yoshihisa

2017-01-01

Aromatic plants are often used as natural medicines because of their remedial and inherent pharmacological properties. Looking into natural resources, particularly products of plant origin, has become an exciting area of research in drug discovery and development. Aromatic plants are mainly exploited for essential oil extraction for applications in industries, for example, in cosmetics, flavoring and fragrance, spices, pesticides, repellents and herbal beverages. Although several medicinal plants have been studied to treat various conventional ailments only a handful studies are available on aromatic plants, especially for radioprotection. Many plant extracts have been reported to contain antioxidants that scavenge free radicals produced due to radiation exposure, thus imparting radioprotective efficacy. The present review focuses on a subset of medicinally important aromatic plants with radioprotective activity. PMID:29134131

[Analysis of aromatic hydrocarbons in cracking products of jet fuel by comprehensive two-dimensional gas chromatography-mass spectrometry].

PubMed

Li, Haijing; Zhang, Xiangwen

2017-08-08

As coking precursors, aromatic hydrocarbons have an effect on the cracking stability of fuels. A method for identifying and quantitating aromatics in the supercritical cracking products of jet fuel was established by comprehensive two-dimensional gas chromatography coupled with mass spectrometry (GC×GC-MS). The effects of main chromatographic conditions such as initial oven temperature and modulation period on the separation of supercritical cracking products were studied. The method has good separation ability for polycyclic aromatic hydrocarbons (PAH) isomers. A total of 27 aromatics, including monocyclic aromatic hydrocarbons, bicyclic aromatic hydrocarbons, tricyclic aromatic hydrocarbons, tetracyclic aromatic hydrocarbons, etc., were identified based on standard mass spectra, the retention times of standards and literature reports. Moreover, the corresponding quantitative determination was achieved by external standard method of GC×GC-FID. The results showed that the contents of aromatics increased with the increase of gas yield. When gas yield reached 22%, the bicyclic aromatic hydrocarbons began to produce, and their contents increased exponentially with the increase of gas yield. Compared with the traditional GC-MS, the method has better separation and qualitative ability, and can be applied to the separation of complex samples and qualitative and quantitative analyses of cracking products.

Chemically bonded stationary phases that use synthetic hosts containing aromatic binding clefts: HPLC analysis of nitro-substituted polycyclic aromatic hydrocarbons.

PubMed Central

Zimmerman, S C; Saionz, K W; Zeng, Z

1993-01-01

The synthesis of hosts with improved binding affinities for nitroaromatic guests is described. Association constants for several host-guest complexes were measured in chloroform solution and ranged over three orders of magnitude. Two hosts were covalently linked to silica gel to produce chemically bonded stationary phases for HPLC. The use of these phases for HPLC analysis of nitro-substituted polycyclic aromatic hydrocarbons is discussed. PMID:8433981

Disseminins and Spiciferone Analogues: Polyketide-Derived Metabolites from a Fungicolous Isolate of Pestalotiopsis disseminata.

PubMed

Hwang, In Hyun; Swenson, Dale C; Gloer, James B; Wicklow, Donald T

2016-03-25

Seven new polyketide metabolites (disseminins A-E, 1-5, and spiciferones D and E, 7 and 8) were obtained from cultures of a fungicolous isolate of Pestalotiopsis disseminata (NRRL 62562), together with a related compound (6) previously known only as a semisynthetic product. Structures were determined mainly by analysis of HRMS and NMR data. Biogenetically related compounds 1 and 2 possess uncommon bis-tetrahydrofuran and dioxabicyclo[3.2.1]octane ring systems, respectively. X-ray crystallographic analysis of the p-bromobenzoate derivative of 1 confirmed the structure and enabled assignment of its absolute configuration.

Model compound study of the pathways for aromatic hydrocarbon formation in soot.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomczyk, N. A.; Hunt, J. E.; Winans, R. E.

2002-04-29

To explore the mechanisms for formation of aromatic hydrocarbons as precursors to soot, a model system using combustion of biphenyl in a fuel rich flame is studied. The soots acquired at three different temperatures are solvent extracted and the extract characterized by both GCMS and high resolution mass spectrometry. A description of the NMR results for the whole soots has been published (1). The production of most products could be rationalized from the coupling of biphenyls and subsequent aromatic species and the addition of acetylenes to existing aromatic molecules. Early work by Badger on pyrolysis of hydrocarbons is used inmore » developing these schemes (2). The reaction schemes to produce larger aromatic hydrocarbons will be discussed. Richter and Howard have discussed in detail potential reaction mechanisms in the formation of aromatics as precursors to soot (3).« less

Investigation of Source of Irritant Gas Produced by PATRIOT Missile System Air Conditioners

DTIC Science & Technology

1986-03-31

is the mass fragment CF3 . It is a common fragment of perfluorinated hydrocarbons, and is found to be present in most of the compounds detected by...used would allow detection of the target par3meters acrolein, aromatics, a broad range of organic compounds ,. formaldehyde, and hydrogen cyanide...organic compounds were observed. Thus, aromatic organic compounds were not produced by or from any of the four new units tested. 4 1CZ 3) With the

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ijam, M.J.; Qatami, S.Y.A.; Arif, S.F.

For several decades removal of aromatics from crude oil fractions (e.g. kerosene and lubricating oils) has been practiced in oil refining to produce fuels and lubricants of lower aromatic content and hence of improved quality. These aromatics are suitable raw materials for the manufacture of aromatic solvents, aromatic process oils, high octane gasoline, and as basic materials for making detergents, perfumes and dyes. Detailed study of molecular structure and substituent effects on the retention characteristics of aromatic hydrocarbons have been reported on alumina, silica and various chemically bonded silicas containing -C/sub 18/, -NH/sub 2/, -R(NH)/sub 2//sub 2/, -CN, RCN, RONmore » and phenyl-mercuric acetate for the compound class (ring-numbered) high performance liquid chromatography (2, 3, 8, 12, 24, 28). Previous work in this laboratory has demonstrated that individual normal and branched aliphatic hydrocarbons from kerosene and light gas oil were isolated and identified. This paper describes the extension of this work to cover the separation and identification of aromatic ring classes (mono-, di-, and tri-aromatics) in the gas oil fraction of Kuwait petroleum. Characterization and identification of the major components in the dinuclear aromatics is our primary objective in this study.« less

[Chemical-genetics based screening for furanonaphthoquinone producing endophytic actinomycetes from seeds of Trewia nudiflora].

PubMed

Li, Fang; Kang, Qianjin; Yao, Xiaoling; Li, Yanyan; Wei, Maolong; Cao, Yong; Lin, Shuangjun; Bai, Linquan; Ma, Wei; Deng, Zixin

2012-04-04

The seeds of Trewia nudiflora containing maytansine (an anticancer agent), was investigated to explore the endophytic actinomycetes diversity and screen for naphthoquinones producing strain. The seeds of Trewia nudiflora were sliced and plated on different selective media after surface sterilization. Clones that looked like actinomycetes were selected, and classified according to the 16S rRNA sequences. Isolated strains were screened for furanonaphthoquinone biosynthesis gene by PCR, and tested for antibacterial and antifungal activity using Staphyloccocusaureus, Pseudomon-asaeruginosa, Bacillus subtilis, Rhizoctoniasolani and Gibberellasaubinetii. LC-MS and NMR were used to determine the structure of candidate compounds. More than 100 endophytic bacteria were isolated. Among them 66 were streptomycetes. FNQ6 (polyketide synthase Type III) and FNQ21 (carboxymuconate cycloisomerase) were only detected in Streptomyces sp. HTZ 27. We got 5 mg pure furanonaphthoquinone (FNQI) from 1 liter Streptomyces sp. HTZ 27 agar fermentation medium. The use of chemical-genetics method increased the efficiency of screening for target compound producing bacteria.

DNA tests for strawberry: mesifurane "sherry" aroma - FaOMT-SI/NO

USDA-ARS?s Scientific Manuscript database

The amazing flavor and texture in strawberries is caused by a complex balance of numerous sugars and aromatic compounds. One of the most important aromatic compounds contributing to the flavor we have come to love in strawberries is mesifurane. Mesifurane produces a sweet sherry-like aroma and incre...

Process for hydrocracking carbonaceous material to provide fuels or chemical feed stock

DOEpatents

Duncan, Dennis A.

1980-01-01

A process is disclosed for hydrocracking coal or other carbonaceous material to produce various aromatic hydrocarbons including benzene, toluene, xylene, ethylbenzene, phenol and cresols in variable relative concentrations while maintaining a near constant maximum temperature. Variations in relative aromatic concentrations are achieved by changing the kinetic severity of the hydrocracking reaction by altering the temperature profile up to and quenching from the final hydrocracking temperature. The relative concentration of benzene to the alkyl and hydroxyl aromatics is increased by imposing increased kinetic severity above that corresponding to constant heating rate followed by immediate quenching at about the same rate to below the temperature at which dehydroxylation and dealkylation reactions appreciably occur. Similarly phenols, cresols and xylenes are produced in enhanced concentrations by adjusting the temperature profile to provide a reduced kinetic severity relative to that employed when high benzene concentrations are desired. These variations in concentrations can be used to produce desired materials for chemical feed stocks or for fuels.

Polycyclic aromatic hydrocarbon degradation by biosurfactant-producing Pseudomonas sp. IR1.

PubMed

Kumara, Manoj; Leon, Vladimir; De Sisto Materano, Angela; Ilzins, Olaf A; Galindo-Castro, Ivan; Fuenmayor, Sergio L

2006-01-01

We characterized a newly isolated bacterium, designated as IR1, with respect to its ability to degrade polycyclic aromatic hydrocarbons (PAHs) and to produce biosurfactants. Isolated IR1 was identified as Pseudomonas putida by analysis of 16S rRNA sequences (99.6% homology). It was capable of utilizing two-, three- and four-ring PAHs but not hexadecane and octadecane as a sole carbon and energy source. PCR and DNA hybridization studies showed that enzymes involved in PAH metabolism were related to the naphthalene dioxygenase pathway. Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by IR1 during growth on both water miscible and immiscible substrates. The biosurfactants lowered the surface tension of medium from 54.9 dN cm(-1) to 35.4 dN cm(-1) and formed a stable and compact emulsion with an emulsifying activity of 74% with diesel oil, when grown on dextrose. These findings indicate that this isolate may be useful for bioremediation of sites contaminated with aromatic hydrocarbons.

Polyketide intermediate mimics as probes for revealing cryptic stereochemistry of ketoreductase domains.

PubMed

Li, Yang; Fiers, William D; Bernard, Steffen M; Smith, Janet L; Aldrich, Courtney C; Fecik, Robert A

2014-12-19

Among natural product families, polyketides have shown the most promise for combinatorial biosynthesis of natural product-like libraries. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase domains of polyketide synthases. This chemical tool-based approach was validated using the ketoreductase of pikromycin module 2 (PikKR2) as a model system. Triketide substrate mimics 12 and 13 were designed to increase stability (incorporating a nonhydrolyzable thioether linkage) and minimize nonessential functionality (truncating the phosphopantetheinyl arm). PikKR2 reduction product identities as well as steady-state kinetic parameters were determined by a combination of LC-MS/MS analysis of synthetic standards and a NADPH consumption assay. The d-hydroxyl product is consistent with bioinformatic analysis and results from a complementary biochemical and molecular biological approach. When compared to widely employed substrates in previous studies, diketide 63 and trans-decalone 64, substrates 12 and 13 showed 2-10 fold lower K(M) values (2.4 ± 0.8 and 7.8 ± 2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the nonreducable enol-tautomer, the k(cat) values were attenuated by as much as 15-336 fold relative to known substrates. This study reveals the high stereoselectivity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-based approach in the study of polyketide ketoreductases.

Polyketide synthases of Diaporthe helianthi and involvement of DhPKS1 in virulence on sunflower.

PubMed

Ruocco, Michelina; Baroncelli, Riccardo; Cacciola, Santa Olga; Pane, Catello; Monti, Maurilia Maria; Firrao, Giuseppe; Vergara, Mariarosaria; Magnano di San Lio, Gaetano; Vannacci, Giovanni; Scala, Felice

2018-01-06

The early phases of Diaporthe helianthi pathogenesis on sunflower are characterized by the production of phytotoxins that may play a role in host colonisation. In previous studies, phytotoxins of a polyketidic nature were isolated and purified from culture filtrates of virulent strains of D. helianthi isolated from sunflower. A highly aggressive isolate (7/96) from France contained a gene fragment of a putative nonaketide synthase (lovB) which was conserved in a virulent D. helianthi population. In order to investigate the role of polyketide synthases in D. helianthi 7/96, a draft genome of this isolate was examined. We were able to find and phylogenetically analyse 40 genes putatively coding for polyketide synthases (PKSs). Analysis of their domains revealed that most PKS genes of D. helianthi are reducing PKSs, whereas only eight lacked reducing domains. Most of the identified PKSs have orthologs shown to be virulence factors or genetic determinants for toxin production in other pathogenic fungi. One of the genes (DhPKS1) corresponded to the previously cloned D. helianthi lovB gene fragment and clustered with a nonribosomal peptide synthetase (NRPS) -PKS hybrid/lovastatin nonaketide like A. nidulans LovB. We used DhPKS1 as a case study and carried out its disruption through Agrobacterium-mediated transformation in the isolate 7/96. D. helianthi DhPKS1 deleted mutants were less virulent to sunflower compared to the wild type, indicating a role for this gene in the pathogenesis of the fungus. The PKS sequences analysed and reported here constitute a new genomic resource that will be useful for further research on the biology, ecology and evolution of D. helianthi and generally of fungal plant pathogens.

Secondary Metabolites Produced during the Germination of Streptomyces coelicolor.

PubMed

Čihák, Matouš; Kameník, Zdeněk; Šmídová, Klára; Bergman, Natalie; Benada, Oldřich; Kofroňová, Olga; Petříčková, Kateřina; Bobek, Jan

2017-01-01

Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor . Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer ( quorum sensing ) and/or play a role in competitive microflora repression ( quorum quenching ) in their nature environments.

Secondary Metabolites Produced during the Germination of Streptomyces coelicolor

PubMed Central

Čihák, Matouš; Kameník, Zdeněk; Šmídová, Klára; Bergman, Natalie; Benada, Oldřich; Kofroňová, Olga; Petříčková, Kateřina; Bobek, Jan

2017-01-01

Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor. Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer (quorum sensing) and/or play a role in competitive microflora repression (quorum quenching) in their nature environments. PMID:29326665

Phenolic Polyketides from the Co-Cultivation of Marine-Derived Penicillium sp. WC-29-5 and Streptomyces fradiae 007

PubMed Central

Wang, Yi; Wang, Liping; Zhuang, Yibin; Kong, Fandong; Zhang, Cuixian; Zhu, Weiming

2014-01-01

Penicillium sp. WC-29-5 was co-cultured with Streptomyces fradiae 007 to produce five natural products (1–3, 4a and 4b) that were isolated and characterized by spectroscopic analysis. Interestingly, these compounds were found to be different from those produced in discrete fungal and bacterial controls. Among these compounds, the absolute configurations of compounds 4a and 4b were determined for the first time by X-ray single crystal diffraction experiments and electronic circular dichroism (ECD) calculations. An evaluation of the cytotoxic activities of these compounds revealed that 4b was moderately cytotoxic towards HL-60 and H1975 tumor cells with IC50 values of 3.73 and 5.73 µM, respectively, whereas compound 4a was only moderately cytotoxic towards H1975 cells with an IC50 value of 3.97 µM. PMID:24714124

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagner, Drew T.; Zeng, Jia; Bailey, Constance B.

In an effort to uncover the structural motifs and biosynthetic logic of the relatively uncharacterized trans-acyltransferase polyketide synthases, we have begun the dissection of the enigmatic dehydrating bimodules common in these enzymatic assembly lines. We report the 1.98 Å resolution structure of a ketoreductase (KR) from the first half of a type A dehydrating bimodule and the 2.22 Å resolution structure of a dehydratase (DH) from the second half of a type B dehydrating bimodule. The KR, from the third module of the bacillaene synthase, and the DH, from the tenth module of the difficidin synthase, possess features not observedmore » in structurally characterized homologs. The DH architecture provides clues for how it catalyzes a unique double dehydration. Correlations between the chemistries proposed for dehydrating bimodules and bioinformatic analysis indicate that type A dehydrating bimodules generally produce an α/β-cis alkene moiety, while type B dehydrating bimodules generally produce an α/β-trans, γ/δ-cis diene moiety.« less

Bacillus amyloliquefaciens ssp. plantarum F11 isolated from Algerian salty lake as a source of biosurfactants and bioactive lipopeptides.

PubMed

Daas, Mohamed Seghir; Acedo, Jeella Z; Rosana, Albert Remus R; Orata, Fabini D; Reiz, Béla; Zheng, Jing; Nateche, Farida; Case, Rebecca J; Kebbouche-Gana, Salima; Vederas, John C

2018-01-01

In this study, we identified a new Bacillus strain isolated from an Algerian salty lake that produces metabolites that are active against Gram-positive and Gram-negative bacteria, as well as fungal pathogens. The draft genome sequence of the strain is presented herein. Genome sequence analysis identified the strain to be B. amyloliquefaciens subspecies plantarum F11, and showed that the strain carries the gene clusters for the production of a number of bioactive and surface-active compounds. These include the lipopeptides surfactin and fengycin, antibacterial polyketides macrolactin and bacillaene, and a putative novel lanthipeptide, among others. Through an activity-guided purification method using hydrophobic interaction chromatographic techniques, we confirmed the ability of the strain to produce fengycin lipopeptides. The identities of the isolated fengycin homologs were ascertained through tandem mass spectrometry. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Phenolic polyketides from the co-cultivation of marine-derived Penicillium sp. WC-29-5 and Streptomyces fradiae 007.

PubMed

Wang, Yi; Wang, Liping; Zhuang, Yibin; Kong, Fandong; Zhang, Cuixian; Zhu, Weiming

2014-04-04

Penicillium sp. WC-29-5 was co-cultured with Streptomyces fradiae 007 to produce five natural products (1-3, 4a and 4b) that were isolated and characterized by spectroscopic analysis. Interestingly, these compounds were found to be different from those produced in discrete fungal and bacterial controls. Among these compounds, the absolute configurations of compounds 4a and 4b were determined for the first time by X-ray single crystal diffraction experiments and electronic circular dichroism (ECD) calculations. An evaluation of the cytotoxic activities of these compounds revealed that 4b was moderately cytotoxic towards HL-60 and H1975 tumor cells with IC₅₀ values of 3.73 and 5.73 µM, respectively, whereas compound 4a was only moderately cytotoxic towards H1975 cells with an IC₅₀ value of 3.97 µM.

The Draft Genome Sequence of Actinokineospora bangkokensis 44EHWT Reveals the Biosynthetic Pathway of the Antifungal Thailandin Compounds with Unusual Butylmalonyl-CoA Extender Units.

PubMed

Greule, Anja; Intra, Bungonsiri; Flemming, Stephan; Rommel, Marcel G E; Panbangred, Watanalai; Bechthold, Andreas

2016-11-23

We report the draft genome sequence of Actinokineospora bangkokensis 44EHW T , the producer of the antifungal polyene compounds, thailandins A and B. The sequence contains 7.45 Mb, 74.1% GC content and 35 putative gene clusters for the biosynthesis of secondary metabolites. There are three gene clusters encoding large polyketide synthases of type I. Annotation of the ORF functions and targeted gene disruption enabled us to identify the cluster for thailandin biosynthesis. We propose a plausible biosynthetic pathway for thailandin, where the unusual butylmalonyl-CoA extender unit is incorporated and results in an untypical side chain.

Novel chlorinated dibenzofurans isolated from the cellular slime mold, Polysphondylium filamentosum, and their biological activities.

PubMed

Kikuchi, Haruhisa; Kubohara, Yuzuru; Nguyen, Van Hai; Katou, Yasuhiro; Oshima, Yoshiteru

2013-08-01

Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster. Copyright © 2013 Elsevier Ltd. All rights reserved.

The cytotoxic mechanism of karlotoxin 2 (KmTx 2) from Karlodinium veneficum (Dinophyceae)

PubMed Central

Deeds, Jonathan R.; Hoesch, Robert E.; Place, Allen R.; Kao, Joseph P.Y.

2015-01-01

This study demonstrates that the polyketide toxin karlotoxin 2 (KmTx 2) produced by Karlodinium veneficum, a dinoflagellate associated with fish kills in temperate estuaries worldwide, alters vertebrate cell membrane permeability. Microfluorimetric and electrophysiological measurements were used to determine that vertebrate cellular toxicity occurs through non-selective permeabilization of plasma membranes, leading to osmotic cell lysis. Previous studies showed that KmTx 2 is lethal to fish at naturally-occurring concentrations measured during fish kills, while sub-lethal doses severely damage gill epithelia. This study provides a mechanistic explanation for the association between K. veneficum blooms and fish kills that has long been observed in temperate estuaries worldwide. PMID:25546005

Pestalpolyols A-D, Cytotoxic Polyketides from Pestalotiopsis sp. cr013.

PubMed

Li, Jing; Xie, Jin; Yang, Yin-He; Li, Xiao-Lian; Zeng, Ying; Zhao, Pei-Ji

2015-09-01

Four novel polyketides, named pestalpolyols A (1), B (2), C (3), and D (4), were isolated from solid fermentation products of Pestalotiopsis sp. cr013. Their structures were elucidated by extensive spectroscopic methods, including 1D and 2D nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry experiments, and the absolute configuration was confirmed by single-crystal X-ray diffraction analysis using the anomalous scattering of Cu Kα radiation. The inhibitory activities of compounds 1, 2, and 4 against five human tumor lines were tested in vitro, and showed IC50 values 2.3-31.2 µM. Georg Thieme Verlag KG Stuttgart · New York.

Forazoline A: Marine-derived polyketide with antifungal in vivo efficacy

DOE PAGES

Wyche, Thomas P.; Piotrowski, Jeff S.; Hou, Yanpeng; ...

2014-09-04

Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine-invertebrate-associated bacteria. Forazoline A had a highly unusual and unprecedented skeleton. Acquisition of 13C– 13C gCOSY and 13C– 15N HMQC NMR data provided the direct carbon–carbon and carbon–nitrogen connectivity, respectively. This approach represents the first example of determining direct 13C– 15N connectivity for a natural product. In this paper, using yeast chemical genomics, we propose that forazoline A operated through a new mechanism of action with a phenotypic outcome of disrupting membrane integrity.

Nonthmicin, a Polyether Polyketide Bearing a Halogen-Modified Tetronate with Neuroprotective and Antiinvasive Activity from Actinomadura sp.

PubMed

Igarashi, Yasuhiro; Matsuoka, Noriaki; In, Yasuko; Kataura, Tetsushi; Tashiro, Etsu; Saiki, Ikuo; Sudoh, Yuri; Duangmal, Kannika; Thamchaipenet, Arinthip

2017-03-17

Nonthmicin (1), a new polyether polyketide bearing a chlorinated tetronic acid, was isolated from the culture extract of a soil-derived Actinomadura strain. The structure of 1 was elucidated by interpretation of NMR and MS spectroscopic data, and the absolute configuration of 1 was proposed on the basis of the crystal structure of its dechloro congener ecteinamycin (2) also isolated from the same strain. Tetronic acids modified by halogenation have never been reported from natural products. Compounds 1 and 2 were found to have neuroprotective activity and antimetastatic properties at submicromolar concentrations in addition to antibacterial activity.

The Post-polyketide Synthase Steps in iso-Migrastatin Biosynthesis Featuring Tailoring Enzymes with Broad Substrate Specificity

PubMed Central

Ma, Ming; Kwong, Thomas; Lim, Si-Kyu; Ju, Jianhua; Lohman, Jeremy R.; Shen, Ben

2013-01-01

The iso-migrastatin (iso-MGS) biosynthetic gene cluster from Streptomyces platensis NRRL 18993 consists of 11 genes, featuring an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes MgsIJK. Systematic inactivation of mgsIJK in S. platensis enabled us to (i) identify two nascent products (10 and 13) of the iso-MGS AT-less type I PKS, establishing an unprecedented novel feature for AT-less type I PKSs, and (ii) account for the formation of all known post-PKS biosynthetic intermediates (10-17) generated by the three tailoring enzymes MgsIJK, which possessed significant substrate promiscuities. PMID:23394593

Characterization of a polyketide synthase in Aspergillus niger whose product is a precursor for both dihydroxynaphthalene (DHN) melanin and naphtho-γ-pyrone.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiang, Yi Ming; Meyer, Kristen M; Praseuth, Michael

2010-12-06

The genome sequencing of the fungus Aspergillus niger, an industrial workhorse, uncovered a large cache of genes encoding enzymes thought to be involved in the production of secondary metabolites yet to be identified. Identification and structural characterization of many of these predicted secondary metabolites are hampered by their low concentration relative to the known A. niger metabolites such as the naphtho-γ-pyrone family of polyketides. We deleted a nonreducing PKS gene in A. niger strain ATCC 11414, a daughter strain of A. niger ATCC strain 1015 whose genome was sequenced by the DOE Joint Genome Institute. This PKS encoding gene ismore » a predicted ortholog of alb1 from Aspergillus fumigatus which is responsible for production of YWA1, a precursor of fungal DHN melanin. Our results show that the A. niger alb1 PKS is responsible for the production of the polyketide precursor for DHN melanin biosynthesis. Deletion of alb1 elimnates the production of major metabolites, naphtho-γ-pyrones. The generation of an A. niger strain devoid of naphtho-γ-pyrones will greatly facilitate the elucidation of cryptic biosynthetic pathways in this organism.« less

Physicochemical and sensory analysis of USA rice varieties developed for the basmati and jasmine markets

USDA-ARS?s Scientific Manuscript database

There is a steady demand for imported basmati and jasmine rice in The USA. Rice varieties that can be domestically produced and compete with these imports, have been developed from basmati, jasmine, and other aromatic germplasm sources. This study evaluated differences among eight USA aromatic varie...

Synthesis of a Fluorescent Acridone Using a Grignard Addition, Oxidation, and Nucleophilic Aromatic Substitution Reaction Sequence

ERIC Educational Resources Information Center

Goodrich, Samuel; Patel, Miloni; Woydziak, Zachary R.

2015-01-01

A three-pot synthesis oriented for an undergraduate organic chemistry laboratory was developed to construct a fluorescent acridone molecule. This laboratory experiment utilizes Grignard addition to an aldehyde, alcohol oxidation, and iterative nucleophilic aromatic substitution steps to produce the final product. Each of the intermediates and the…

Melanin is required for the formation of the multi-cellular conidia in the endophytic fungus Pestalotiopsis microspora.

PubMed

Yu, Xi; Huo, Liang; Liu, Heng; Chen, Longfei; Wang, Yu; Zhu, Xudong

2015-10-01

Melanin plays an important role in regulating various biological processes in many fungi. However, its biological role in conidiation remains largely elusive. We report here that conidia production, morphogenesis, integrity, germination and their viability in Pestalotiopsis microspora require the polyketide-derived melanin. A polyketide synthase gene, pks1, was identified and demonstrated responsible for melanin biosynthesis in this fungus. A targeted deletion mutant strain Δpks1 displayed a defect in pigmentation of conidia and had an albino colonial phenotype. Interestingly, Δpks1 produced approximately 6-fold as many conidia as the wild type did, suggesting a negative modulation of melanin on conidia production in this fungus. Moreover, the conidia failed to develop into the normal five-cell morphology, rather the three main-body cells separated via constriction at the original septum position to generate three independent mutant conidia. This result suggests a novel role of melanin in the formation of the multi-cellular conidia. Germ tubes could develop from the three different types of mutant conidia and kept elongating, despite a significantly lower germination rate was observed for them. Still more, the unpigmented conidia became permeable to Calcofluor White and DAPI, suggesting the integrity of the conidia was impaired. Deliberate inhibition of melanin biosynthesis by a specific inhibitor, tricyclazole, led to a similar phenotypes. This work demonstrates a new function of fungal melanin in conidial development. Copyright © 2015 Elsevier GmbH. All rights reserved.

Genome-guided exploration of metabolic features of Streptomyces peucetius ATCC 27952: past, current, and prospect.

PubMed

Thuan, Nguyen Huy; Dhakal, Dipesh; Pokhrel, Anaya Raj; Chu, Luan Luong; Van Pham, Thi Thuy; Shrestha, Anil; Sohng, Jae Kyung

2018-05-01

Streptomyces peucetius ATCC 27952 produces two major anthracyclines, doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for the treatment of several cancers. In order to gain detailed insight on genetics and biochemistry of the strain, the complete genome was determined and analyzed. The result showed that its complete sequence contains 7187 protein coding genes in a total of 8,023,114 bp, whereas 87% of the genome contributed to the protein coding region. The genomic sequence included 18 rRNA, 66 tRNAs, and 3 non-coding RNAs. In silico studies predicted ~ 68 biosynthetic gene clusters (BCGs) encoding diverse classes of secondary metabolites, including non-ribosomal polyketide synthase (NRPS), polyketide synthase (PKS I, II, and III), terpenes, and others. Detailed analysis of the genome sequence revealed versatile biocatalytic enzymes such as cytochrome P450 (CYP), electron transfer systems (ETS) genes, methyltransferase (MT), glycosyltransferase (GT). In addition, numerous functional genes (transporter gene, SOD, etc.) and regulatory genes (afsR-sp, metK-sp, etc.) involved in the regulation of secondary metabolites were found. This minireview summarizes the genome-based genome mining (GM) of diverse BCGs and genome exploration (GE) of versatile biocatalytic enzymes, and other enzymes involved in maintenance and regulation of metabolism of S. peucetius. The detailed analysis of genome sequence provides critically important knowledge useful in the bioengineering of the strain or harboring catalytically efficient enzymes for biotechnological applications.

Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome.

PubMed

Müller, Christina A; Oberauner-Wappis, Lisa; Peyman, Armin; Amos, Gregory C A; Wellington, Elizabeth M H; Berg, Gabriele

2015-08-01

Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Inhibition of hydroxycinnamoyl-CoA thioesterases in ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.) by lipase inhibitors.

PubMed

Flores-Sanchez, Isvett Josefina; Gang, David Roger

2013-11-01

Ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.), members of the Zingiberaceae, are widely used in traditional Asian cuisines and herbal medicine. Gingerols and diarylheptanoids, important compounds from these plants, appear to be produced by enzymes of the type III polyketide synthase class. Previous efforts to detect activity of such enzymes in tissues from these plants were only marginally successful in turmeric and completely unsuccessful in ginger because of very rapid hydrolysis of the hydroxycinnamoyl-CoA substrates (p-coumaroyl-CoA, feruloyl-CoA and caffeoyl-CoA) in these assays, presumably due to the presence of thioesterases in these tissues. In order to determine whether such thioesterase activities were specific and could be reduced so that the polyketide synthase activities could be better characterized, three inhibitors of the thioesterase domain of fatty acid synthase were tested in assays with leaf and rhizome crude protein extracts from these plants: orlistat, a reduced form of lipstatin, and peptide 1 and peptide 2 from hydrolysates of soybean β-conglycinin. Results of these analyses indicated that specific thioesterases do exist in these plants and that they could indeed be inhibited, with highest inhibition occurring with a mixture of these three compounds, leading for example to a reduction of caffeoyl-CoA hydrolysis in leaves and rhizomes of ginger by 40-fold and 27-fold, respectively. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yung, Matthew M.; Stanton, Alexander R.; Iisa, Kristiina

Metal-impregnated (Ni or Ga) ZSM-5 catalysts were studied for biomass pyrolysis vapor upgrading to produce hydrocarbons using three reactors constituting a 100 000x change in the amount of catalyst used in experiments. Catalysts were screened for pyrolysis vapor phase upgrading activity in two small-scale reactors: (i) a Pyroprobe with a 10 mg catalyst in a fixed bed and (ii) a fixed-bed reactor with 500 mg of catalyst. The best performing catalysts were then validated with a larger scale fluidized-bed reactor (using ~1 kg of catalyst) that produced measurable quantities of bio-oil for analysis and evaluation of mass balances. Despite somemore » inherent differences across the reactor systems (such as residence time, reactor type, analytical techniques, mode of catalyst and biomass feed) there was good agreement of reaction results for production of aromatic hydrocarbons, light gases, and coke deposition. Relative to ZSM-5, Ni or Ga addition to ZSM-5 increased production of fully deoxygenated aromatic hydrocarbons and light gases. In the fluidized bed reactor, Ga/ZSM-5 slightly enhanced carbon efficiency to condensed oil, which includes oxygenates in addition to aromatic hydrocarbons, and reduced oil oxygen content compared to ZSM-5. Ni/ZSM-5, while giving the highest yield of fully deoxygenated aromatic hydrocarbons, gave lower overall carbon efficiency to oil but with the lowest oxygen content. Reaction product analysis coupled with fresh and spent catalyst characterization indicated that the improved performance of Ni/ZSM-5 is related to decreasing deactivation by coking, which keeps the active acid sites accessible for the deoxygenation and aromatization reactions that produce fully deoxygenated aromatic hydrocarbons. The addition of Ga enhances the dehydrogenation activity of the catalyst, which leads to enhanced olefin formation and higher fully deoxygenated aromatic hydrocarbon yields compared to unmodified ZSM-5. Catalyst characterization by ammonia temperature programmed desorption, surface area measurements, and postreaction temperature-programmed oxidation (TPO) also showed that the metal-modified zeolites retained a greater percentage of their initial acidity and surface area, which was consistent between the reactor scales. These results demonstrate that the trends observed with smaller (milligram to gram) catalyst reactors are applicable to larger, more industrially relevant (kg) scales to help guide catalyst research toward application.« less

δ 13C of free and macromolecular aromatic structures in the murchison meteorite

NASA Astrophysics Data System (ADS)

Sephton, M. A.; Pillinger, C. T.; Gilmour, I.

1998-05-01

Analyses of the organic compounds in the Murchison meteorite have led to a greater understanding of the nature of extraterrestrial organic materials. However, the relationship between low and high molecular weight material remains poorly understood. To investigate this relationship, untreated Murchison was subjected to supercritical fluid extraction (SFE) to obtain the free organic components in the meteorite. Toluene and other volatile aromatic hydrocarbons dominated the extract, and the carbon isotopic composition of these molecules was determined by gas chromatography-isotope ratio-mass spectrometry (GCIRMS). δ 13C values of the aromatic hydrocarbons ranged from -28.8 to -5.8‰. These compounds displayed a 13C-enrichment with increasing carbon number suggesting an origin by cracking. The high molecular weight organic material in the meteorite was isolated and subjected to hydrous pyrolysis. This procedure produced a number of aromatic products, the majority of which were volatile aromatic hydrocarbons, particularly toluene. SFE was used to extract and successfully retain them. This enabled the first carbon isotopic analysis of this poorly understood material to be performed at the molecular level by GCIRMS. δ 13C values for aromatic pyrolysis products occupied a range from -24.6 to -5.6‰. The trend of 13C-enrichment with increasing carbon number, observed in the free compounds, was also evident in the macromolecular fragments. Furthermore, the organic fragments of the macromolecular material were consistently 13C-enriched when compared to structurally identical free molecules. This suggested that the free aromatic hydrocarbons in Murchison were produced by the preterrestrial degradation of the organic macromolecular material. This natural degradation event was extended by the hydrous pyrolysis experiment.

Pseudomonas putida as a platform for the synthesis of aromatic compounds.

PubMed

Molina-Santiago, Carlos; Cordero, Baldo F; Daddaoua, Abdelali; Udaondo, Zulema; Manzano, Javier; Valdivia, Miguel; Segura, Ana; Ramos, Juan-Luis; Duque, Estrella

2016-09-01

Aromatic compounds such as l-phenylalanine, 2-phenylethanol and trans-cinnamate are aromatic compounds of industrial interest. Current trends support replacement of chemical synthesis of these compounds by 'green' alternatives produced in microbial cell factories. The solvent-tolerant Pseudomonas putida DOT-T1E strain was genetically modified to produce up to 1 g l-1 of l-phenylalanine. In order to engineer this strain, we carried out the following stepwise process: (1) we selected random mutants that are resistant to toxic phenylalanine analogues; (2) we then deleted up to five genes belonging to phenylalanine metabolism pathways, which greatly diminished the internal metabolism of phenylalanine; and (3) in these mutants, we overexpressed the pheAfbr gene, which encodes a recombinant variant of PheA that is insensitive to feedback inhibition by phenylalanine. Furthermore, by introducing new genes, we were able to further extend the diversity of compounds produced. Introduction of histidinol phosphate transferase (PP_0967), phenylpyruvate decarboxylase (kdc) and an alcohol dehydrogenase (adh) enabled the strain to produce up to 180 mg l-1 2-phenylethanol. When phenylalanine ammonia lyase (pal) was introduced, the resulting strain produced up to 200 mg l-1 of trans-cinnamate. These results demonstrate that P. putida can serve as a promising microbial cell factory for the production of l-phenylalanine and related compounds.

Organic solids produced by electrical discharges in reducing atmospheres: Molecular analysis

NASA Technical Reports Server (NTRS)

Khare, B. N.; Sagan, C.; Zumberge, J. E.; Sklarew, D.; Nagy, B.

1978-01-01

The complex brown polymer produced on passage of an electrical discharge through a mixture of methane, ammonia, and water, is analyzed by pyrolytic GC/MS. Pyrolyzates include a wide range of alkanes, alkenes, aromatic hydrocarbons, aliphatic and aromatic nitriles, pyrroles, and pyridine. Similar pyrolyzates are obtained from polypeptides and polynucleotides with hydrocarbon moieties. This polymer is remarkably stable up to 950 C; its degradation products are candidate constituents of planetary aerosols in the outer solar system and the grains and gas in the interstellar medium.

Riboflavin induces Metarhizium spp. to produce conidia with elevated tolerance to UV-B, and upregulates photolyases, laccases and polyketide synthases genes.

PubMed

Pereira-Junior, R A; Huarte-Bonnet, C; Paixão, F R S; Roberts, D W; Luz, C; Pedrini, N; Fernandes, É K K

2018-02-23

The effect of nutritional supplementation of two Metarhizium species with riboflavin (Rb) during production of conidia was evaluated on (i) conidial tolerance (based on germination) to UV-B radiation and on (ii) conidial expression following UV-B irradiation, of enzymes known to be active in photoreactivation, viz., photolyase (Phr), laccase (Lac) and polyketide synthase (Pks). Metarhizium acridum (ARSEF 324) and Metarhizium robertsii (ARSEF 2575) were grown either on (i) potato dextrose agar medium (PDA), (ii) PDA supplemented with 1% yeast extract (PDAY), (iii) PDA supplemented with Rb (PDA+Rb), or (iv) PDAY supplemented with Rb (PDAY+Rb). Resulting conidia were exposed to 866·7 mW m -2 of UV-B Quaite-weighted irradiance to total doses of 3·9 or 6·24 kJ m -2 . Some conidia also were exposed to 16 klux of white light (WL) after being irradiated, or not, with UV-B to investigate the role of possible photoreactivation. Relative germination of conidia produced on PDA+Rb (regardless Rb concentration) or on PDAY and exposed to UV-B was higher compared to conidia cultivated on PDA without Rb supplement, or to conidia suspended in Rb solution immediately prior to UV-B exposure. The expression of MaLac3 and MaPks2 for M. acridum, as well as MrPhr2, MrLac1, MrLac2 and MrLac3 for M. robertsii was higher when the isolates were cultivated on PDA+Rb and exposed to UV-B followed by exposure to WL, or exposed to WL only. Rb in culture medium increases the UV-B tolerance of M. robertsii and M. acridum conidia, and which may be related to increased expression of Phr, Lac and Pks genes in these conidia. The enhanced UV-B tolerance of Metarhizium spp. conidia produced on Rb-enriched media may improve the effectiveness of these fungi in biological control programs. © 2018 The Society for Applied Microbiology.

The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110

PubMed Central

2012-01-01

Background Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. Results Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element. Conclusions The complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest. PMID:22443545

The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110.

PubMed

Schwientek, Patrick; Szczepanowski, Rafael; Rückert, Christian; Kalinowski, Jörn; Klein, Andreas; Selber, Klaus; Wehmeier, Udo F; Stoye, Jens; Pühler, Alfred

2012-03-23

Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element. The complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest.

Which kind of aromatic structures are produced during biomass charring? New insights provided by modern solid-state NMR spectroscopy

NASA Astrophysics Data System (ADS)

Knicker, Heike; Paneque-Carmona, Marina; Velasco-Molina, Marta; de la Rosa, José Maria; León-Ovelar, Laura Regina; Fernandez-Boy, Elena

2017-04-01

Intense research on biochar and charcoal of the last years has revealed that depending on the production conditions, the chemical and physical characteristics of their aromatic network can greatly vary. Since such variations are determining the behavior and stability of charred material in soils, a better understanding of the structural changes occurring during their heating and the impact of those changes on their function is needed. One method to characterize pyrogenic organic matter (PyOM) represents solid-state 13C NMR spectroscopy applying the cross polarization (CP) magic angle spinning technique (MAS). A drawback of this technique is that the quantification of NMR spectra of samples with highly condensed and proton-depleted structures is assumed to be bias. Typical samples with such attributes are charcoals produced at temperatures above 700°C under pyrolytic conditions. Commonly their high condensation degree leads to graphenic structures that are not only reducing the CP efficiency but create also a conductive lattice which acts as a shield and prevents the entering of the excitation pulse into the sample during the NMR experiments. Since the latter can damage the NMR probe and in the most cases the obtained NMR spectra show only one broad signal assignable to aromatic C, this technique is rarely applied for characterizing high temperature chars or soot. As a consequence, a more detailed knowledge of the nature of the aromatic ring systems is still missing. The latter is also true for the aromatic domains of PyOM produced at lower temperatures, since older NMR instruments operating at low magnetic fields deliver solid-state 13C NMR spectra with low resolution which turns a more detailed analysis of the aromatic chemical shift region into a challenging task. In order to overcome this disadvantages, modern NMR spectroscopy offers not only instruments with greatly improved resolution but also special pulse sequences for NMR experiments which allow a more detailed chemical shift assignment. Applying the latter to various charcoals and biochars, we intended to test their usefulness for a better characterization of PyOM and elucidation how specific aromatic features can affect their behavior in soils. We could demonstrate that furans represent the major compound class of low temperature chars produced from woody material. As indicated by 2D techniques, residual alkyl C in such chars has minor covalent binding to the aromatic network. Reducing the electrical conductivity of high-temperature chars by addition of aluminum oxide permitted the application of the cross CP technique. Determination of the relaxation and CP dynamics confirmed high rigidity of their aromatic domains which were dominated by coronene-type moieties. In contrast to common view, we could demonstrate that quantifiable CP NMR spectra can be obtained from high temperature chars with contact times of 3 to 5 ms and pulse delays > 3 s.

Effects of petroleum hydrocarbons on hepatic function in the duck

USGS Publications Warehouse

Patton, J.F.; Dieter, M.P.

1980-01-01

1. The indocyanine green dye clearance test for hepatic function was determined in mallard ducks before and during the chronic ingestion (7 months) of representative paraffinic or aromatic petroleum hydrocarbons (PH).2. No mortality or visible symptoms of toxicity occured in any of the tests. Ingestion of 4000 ppm aromatic PH produced significant increases in liver (25%), plasma clearance of indocyanine green (33%) and hepatic blood flow (30%).3. Although the aromatics elicited a greater hepatic stress response than the paraffins, the ducks tolerated high concentrations of PH for extended periods.

Polyphenylquinoxalines via Aromatic Nucleophilic Displacement

NASA Technical Reports Server (NTRS)

Hergenrother, Paul M.; Connell, John W.

1988-01-01

Polyphenylquinoxalines are produced by an aromatic nucleophilic displacement reaction involving an activated aromatic dihalide with an appropriate quinoxaline monomer. Polyphenylquinoxalines are high temperature thermoplastics used as adhesives, coatings, films and composite matrices. The novelty of this invention is threefold: (1) some of the quinoxaline monomers are new compositions of matter; (2) the phenylquinoxaline polymers which are the end products of the invention are new compositions of matter; and (3) the method of forming the polymers is novel, replacing a more costly prior art process, which is also limited in the kinds of products prepared therefrom.

Airborne Exposures to Polycyclic Aromatic Compounds Among Workers in Asphalt Roofing Manufacturing Facilities.

PubMed

Trumbore, David C; Osborn, Linda V; Johnson, Kathleen A; Fayerweather, William E

2015-01-01

We studied exposure of 151 workers to polycyclic aromatic compounds and asphalt emissions during the manufacturing of asphalt roofing products-including 64 workers from 10 asphalt plants producing oxidized, straight-run, cutback, and wax- or polymer-modified asphalts, and 87 workers from 11 roofing plants producing asphalt shingles and granulated roll roofing. The facilities were located throughout the United States and used asphalt from many refiners and crude oils. This article helps fill a gap in exposure data for asphalt roofing manufacturing workers by using a fluorescence technique that targets biologically active 4-6 ring polycyclic aromatic compounds and is strongly correlated with carcinogenic activity in animal studies. Worker exposures to polycyclic aromatic compounds were compared between manufacturing plants, at different temperatures and using different raw materials, and to important external benchmarks. High levels of fine limestone particulate in the plant air during roofing manufacturing increased polycyclic aromatic compound exposure, resulting in the hypothesis that the particulate brought adsorbed polycyclic aromatic compounds to the worker breathing zone. Elevated asphalt temperatures increased exposures during the pouring of asphalt. Co-exposures in these workplaces which act as confounders for both the measurement of total organic matter and fluorescence were detected and their influence discussed. Exposures to polycyclic aromatic compounds in asphalt roofing manufacturing facilities were lower than or similar to those reported in hot-mix paving application studies, and much below those reported in studies of hot application of built-up roofing asphalt. These relatively low exposures in manufacturing are primarily attributed to air emission controls in the facilities, and the relatively moderate temperatures, compared to built-up roofing, used in these facilities for oxidized asphalt. The exposure to polycyclic aromatic compounds was a very small part of the overall worker exposure to asphalt fume, on average less than 0.07% of the benzene-soluble fraction. Measurements of benzene-soluble fraction were uniformly below the American Conference of Governmental Industrial Hygienists' Threshold Limit Value for asphalt fume.

Molecular screening of xerophilic Aspergillus strains producing mycophenolic acid.

PubMed

Mouhamadou, Bello; Sage, Lucile; Périgon, Sophie; Séguin, Virginie; Bouchart, Valérie; Legendre, Patrick; Caillat, Mathilde; Yamouni, Hayet; Garon, David

2017-02-01

Mycophenolic acid (MPA) is the fungal secondary metabolite displaying several biological properties. Up to now, screening of fungal strains producing MPA has mainly been the result of the search of this molecule in their culture medium by chemical methods. Here we developed a molecular approach by targeting the expression level of the MpaC gene encoding the polyketide synthase, one of the key enzymes involved in the MPA synthesis. Thirty xerophilic Aspergillus strains were identified using the RNA polymerase II subunit and the β-tubulin genes. Seven Aspergillus species were evidenced. The expression level of the MpaC gene was quantified and compared to the MPA production rate. Only Aspergillus pseudoglaucus and all the eight strains of this species produced MPA. While the MpaC gene was not expressed or weakly expressed in the MPA non-producing strains, all the A. pseudoglaucus strains presented a high level of expression of this gene. The highest expression level of the MpaC gene among the MPA non-producing strains was significantly lower than the lowest expression level of this gene in the MPA producing strains. To our knowledge, this is the first study that demonstrates the effectiveness of molecular approach for the screening of MPA-producing species. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Production of aromatic hydrocarbons via catalytic pyrolysis of biomass over fe-modified HZSM-5 zeolites

USDA-ARS?s Scientific Manuscript database

Iron modified HZSM-5 catalysts were prepared by partial ion exchange of NH4ZSM-5 with Fe (II) at three different loadings (1.4, 2.8 and 4.2 wt%), and their effectiveness for producing aromatic hydrocarbons from cellulose, cellobiose, lignin and switchgrass by catalytic pyrolysis were screened using ...

The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

USDA-ARS?s Scientific Manuscript database

The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2, which encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rod...

From Biomass-Derived Furans to Aromatics with Ethanol over Zeolite.

PubMed

Teixeira, Ivo F; Lo, Benedict T W; Kostetskyy, Pavlo; Stamatakis, Michail; Ye, Lin; Tang, Chiu C; Mpourmpakis, Giannis; Tsang, Shik Chi Edman

2016-10-10

We report a novel catalytic conversion of biomass-derived furans and alcohols to aromatics over zeolite catalysts. Aromatics are formed via Diels-Alder cycloaddition with ethylene, which is produced in situ from ethanol dehydration. The use of liquid ethanol instead of gaseous ethylene, as the source of dienophile in this one-pot synthesis, makes the aromatics production much simpler and renewable, circumventing the use of ethylene at high pressure. More importantly, both our experiments and theoretical studies demonstrate that the use of ethanol instead of ethylene, results in significantly higher rates and higher selectivity to aromatics, due to lower activation barriers over the solid acid sites. Synchrotron-diffraction experiments and proton-affinity calculations clearly suggest that a preferred protonation of ethanol over the furan is a key step facilitating the Diels-Alder and dehydration reactions in the acid sites of the zeolite. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Physical and chemical characterization of representative samples of recycled rubber from end-of-life tires.

PubMed

Re Depaolini, Andrea; Bianchi, Giancarlo; Fornai, Daniele; Cardelli, Angela; Badalassi, Marco; Cardelli, Camillo; Davoli, Enrico

2017-10-01

A large number of end-of-life tires (ELTs) were sampled and classified by type, age and origin to obtain recycled rubber samples representative of the materials placed on the Italian market. The selected recycled tire rubber samples were physically and chemically characterized and a chemometric approach was used to determine correlations. The polycyclic aromatic hydrocarbons (PAHs) content was correlated to the aromaticity index and a model was built to establish the H-Bay aromaticity index (H-Bay) from the PAH concentrations. ELT of different origin and age produced in non-European countries generally had higher PAH content and a higher H-Bay index. H-Bay values of all the samples were lower than the REACH limits and old tires had higher aromatic content than recent ones, possibly due to the replacement of aromatic oils in tire production. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Synthesis of ketones from biomass-derived feedstock.

PubMed

Meng, Qinglei; Hou, Minqiang; Liu, Huizhen; Song, Jinliang; Han, Buxing

2017-01-31

Cyclohexanone and its derivatives are very important chemicals, which are currently produced mainly by oxidation of cyclohexane or alkylcyclohexane, hydrogenation of phenols, and alkylation of cyclohexanone. Here we report that bromide salt-modified Pd/C in H 2 O/CH 2 Cl 2 can efficiently catalyse the transformation of aromatic ethers, which can be derived from biomass, to cyclohexanone and its derivatives via hydrogenation and hydrolysis processes. The yield of cyclohexanone from anisole can reach 96%, and the yields of cyclohexanone derivatives produced from the aromatic ethers, which can be extracted from plants or derived from lignin, are also satisfactory. Detailed study shows that the Pd, bromide salt and H 2 O/CH 2 Cl 2 work cooperatively to promote the desired reaction and inhibit the side reaction. Thus high yields of desired products can be obtained. This work opens the way for production of ketones from aromatic ethers that can be derived from biomass.

Improving the hydrocarbon production via co-pyrolysis of bagasse with bio-plastic and dual-catalysts layout.

PubMed

Zhang, Huiyan; Likun, Peter Keliona Wani; Xiao, Rui

2018-03-15

Catalytic fast pyrolysis (CFP) of bagasse and bio-plastic (chicken feather keratin) and their mixtures were conducted to produce aromatic hydrocarbons over a HZSM-5, USY, and dual-catalysts layout. The effects of temperature, co-feeding ratios, feed-to-catalyst ratios and dual catalysts on hydrocarbon yields and selectivities were investigated. The results show a general improvement in the aromatic hydrocarbons yields in all cases compared to non-catalytic and pure biomass pyrolysis. The aromatic hydrocarbons increased by 10 fold with the increase of temperature from 400°C to 700°C. The aromatic yields increased 1.5 times at co-feeding, 2.0 greater at feed/HZSM-5 ratio of 1:6, 1.2 times at feed/USY ratio of 1:16, and 2.66 times at USY/HZSM-5 scenario. The selectivities towards benzene increased, at higher co-feeding ratios, while that of toluene shows an opposite trend. Xylenes selectivities were less sensitive to the changes of co-feeding ratios. In contrast, the USY catalyst only produced little amount of toluene and xylenes. The dual catalyst design (USY/HZSM-5) resulted in the highest aromatic yields, than other catalyst design scenarios. The pyrolysis temperature is a significant parameter for hydrocarbon production. Co-feeding bagasse and bio-plastic enhanced biomass conversion to aromatic compounds. For any type of zeolite catalyst, there was an optimum feed-to-catalyst ratio that generated maximum hydrocarbons. Dual catalyst layout shows a new opportunity for efficient conversion of biomass materials into hydrocarbons. Copyright © 2017 Elsevier B.V. All rights reserved.

Lignin solubilization and aqueous phase reforming for the production of aromatic chemicals and hydrogen.

PubMed

Zakzeski, Joseph; Weckhuysen, Bert M

2011-03-21

The solubilization and aqueous phase reforming of lignin, including kraft, soda, and alcell lignin along with sugarcane bagasse, at low temperatures (T≤498 K) and pressures (P≤29 bar) is reported for the first time for the production of aromatic chemicals and hydrogen. Analysis of lignin model compounds and the distribution of products obtained during the lignin aqueous phase reforming revealed that lignin was depolymerized through disruption of the abundant β-O-4 linkages and, to a lesser extent, the 5-5' carbon-carbon linkages to form monomeric aromatic compounds. The alkyl chains contained on these monomeric compounds were readily reformed to produce hydrogen and simple aromatic platform chemicals, particularly guaiacol and syringol, with the distribution of each depending on the lignin source. The methoxy groups present on the aromatic rings were subject to hydrolysis to form methanol, which was also readily reformed to produce hydrogen and carbon dioxide. The composition of the isolated yields of monomeric aromatic compounds and overall lignin conversion based on these isolated yields varied from 10-15% depending on the lignin sample, with the balance consisting of gaseous products and residual solid material. Furthermore, we introduce the use of a high-pressure autoclave with optical windows and an autoclave with ATR-IR sentinel for on-line in situ spectroscopic monitoring of biomass conversion processes, which provides direct insight into, for example, the solubilization process and aqueous phase reforming reaction of lignin. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Confluence of structural and chemical biology: plant polyketide synthases as biocatalysts for a bio-based future.

PubMed

Stewart, Charles; Vickery, Christopher R; Burkart, Michael D; Noel, Joseph P

2013-06-01

Type III plant polyketide synthases (PKSs) biosynthesize a dazzling array of polyphenolic products that serve important roles in both plant and human health. Recent advances in structural characterization of these enzymes and new tools from the field of chemical biology have facilitated exquisite probing of plant PKS iterative catalysis. These tools have also been used to exploit type III PKSs as biocatalysts to generate new chemicals. Going forward, chemical, structural and biochemical analyses will provide an atomic resolution understanding of plant PKSs and will serve as a springboard for bioengineering and scalable production of valuable molecules in vitro, by fermentation and in planta. Copyright © 2013 Elsevier Ltd. All rights reserved.

A "methyl extension" strategy for polyketide natural product linker site validation and its application to dictyostatin.

PubMed

Ho, Stephen; Sackett, Dan L; Leighton, James L

2015-11-11

An approach to the validation of linker strategies for polyketide natural products with few or no obvious handles for linker attachment, and its application to dictyostatin, are described. Analogues in which the C(6)- and C(12)-methyl groups were replaced by 4-azidobutyl groups were prepared and shown to retain the low nanomolar potency of dictyostatin. Further, conjugation of the C(6) analogue with a cyclooctyne resulted in only minor attenuations in potency. Together, these results shed light on the binding of dictyostatin to β-tubulin, establish a validated linker strategy for dictyostatin, and set the stage for the synthesis and study of dictyostatin conjugates.

Heterogeneous Diels–Alder catalysis for biomass-derived aromatic compounds

DOE PAGES

Settle, Amy E.; Berstis, Laura; Rorrer, Nicholas A.; ...

2017-05-17

In this tutorial review, we provide an overview of heterogeneous Diels–Alder catalysis for the production of lignocellulosic biomass-derived aromatic compounds. Diels–Alder reactions afford an extremely selective and efficient route for carbon–carbon cycloadditions to produce intermediates that can readily undergo subsequent dehydration or dehydrogenation reactions for aromatization. As a result, catalysis of Diels–Alder reactions with biomass-derived dienes and dienophiles has seen a growth of interest in recent years; however, significant opportunities remain to (i) tailor heterogeneous catalyst materials for tandem Diels–Alder and aromatization reactions, and (ii) utilize biomass-derived dienes and dienophiles to access both conventional and novel aromatic monomers. As such,more » this review discusses the mechanistic aspects of Diels–Alder reactions from both an experimental and computational perspective, as well as the synergy of Brønsted–Lewis acid catalysts to facilitate tandem Diels–Alder and aromatization reactions. Heterogeneous catalyst design strategies for Diels–Alder reactions are reviewed for two exemplary solid acid catalysts, zeolites and polyoxometalates, and recent efforts for targeting direct replacement aromatic monomers from biomass are summarized. In conclusion, we point out important research directions for progressing Diels–Alder catalysis to target novel, aromatic monomers with chemical functionality that enables new properties compared to monomers that are readily accessible from petroleum.« less

Organic solids produced by electrical discharge in reducing atmospheres - Tholin molecular analysis

NASA Technical Reports Server (NTRS)

Khare, B. N.; Sagan, C.; Zumberge, J. E.; Sklarew, D. S.; Nagy, B.

1981-01-01

The complex dark brown solid of a class called tholins, produced on passage of an electrical discharge through a roughly equimolar mixture of methane and ammonia with 2.6% water vapor, is analyzed by vacuum pyrolysis followed by gas chromatography and mass spectrometry. Pyrolyzates include a wide range of aliphatic and aromatic nitriles, alkanes, alkenes, aromatic hydrocarbons, pyrrole, and pyridine. This tholin is remarkably stable to 950 C. It and its degradation products are candidate constituents of planetary aerosols in the outer solar system and of the grains in the interstellar medium.

Marketable transport fuels made from Julia Creek shale oil

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1987-03-01

CSR Limited and the CSIRO Division of Energy Chemistry have been working on the problem of producing refined products from the Julia Creek deposit in Queensland, Australia. Two samples of shale oil, retorted at different temperatures from Julia Creek oil shale, were found to differ markedly in aromaticity. Using conventional hydrotreating technology, high quality jet and diesel fuels could be made from the less aromatic oil. Naphtha suitable for isomerization and reforming to gasoline could be produced from both oils. This paper discusses oil properties, stabilization of topped crudes, second stage hydrotreatment, and naphtha hydrotreating. 1 figure, 4 tables.

Tough, high performance, addition-type thermoplastic polymers

NASA Technical Reports Server (NTRS)

Pater, Ruth H. (Inventor)

1992-01-01

A tough, high performance polyimide is provided by reacting a triple bond conjugated with an aromatic ring in a bisethynyl compound with the active double bond in a compound containing a double bond activated toward the formation of a Diels-Adler type adduct, especially a bismaleimide, a biscitraconimide, or a benzoquinone, or mixtures thereof. Addition curing of this product produces a high linear polymeric structure and heat treating the highly linear polymeric structure produces a thermally stable aromatic addition-type thermoplastic polyimide, which finds utility in the preparation of molding compounds, adhesive compositions, and polymer matrix composites.

ClusterCAD: a computational platform for type I modular polyketide synthase design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eng, Clara H.; Backman, Tyler W H; Bailey, Constance B.

Here, we present ClusterCAD, a web-based toolkit designed to leverage the collinear structure and deterministic logic of type I modular polyketide synthases (PKSs) for synthetic biology applications. The unique organization of these megasynthases, combined with the diversity of their catalytic domain building blocks, has fueled an interest in harnessing the biosynthetic potential of PKSs for the microbial production of both novel natural product analogs and industrially relevant small molecules. However, a limited theoretical understanding of the determinants of PKS fold and function poses a substantial barrier to the design of active variants, and identifying strategies to reliably construct functional PKSmore » chimeras remains an active area of research. In this work, we formalize a paradigm for the design of PKS chimeras and introduce ClusterCAD as a computational platform to streamline and simplify the process of designing experiments to test strategies for engineering PKS variants. ClusterCAD provides chemical structures with stereochemistry for the intermediates generated by each PKS module, as well as sequence- and structure-based search tools that allow users to identify modules based either on amino acid sequence or on the chemical structure of the cognate polyketide intermediate. ClusterCAD can be accessed at https://clustercad.jbei.org and at http://clustercad.igb.uci.edu.« less

The secreted polyketide boydone A is responsible for the anti-Staphylococcus aureus activity of Scedosporium boydii.

PubMed

Staerck, Cindy; Landreau, Anne; Herbette, Gaëtan; Roullier, Catherine; Bertrand, Samuel; Siegler, Benjamin; Larcher, Gérald; Bouchara, Jean-Philippe; Fleury, Maxime J J

2017-12-01

Usually living as a soil saprophyte, the filamentous fungus Scedosporium boydii may also cause various infections in human. Particularly, it is one of the major causative agents of fungal colonization of the airways in patients with cystic fibrosis (CF). To compete with other microorganisms in the environment, fungi have evolved sophisticated strategies, including the production of secondary metabolites with antimicrobial activity that may also help them to establish successfully within the respiratory tract of receptive hosts. Here, the culture filtrate from a human pathogenic strain of S. boydii was investigated searching for an antibacterial activity, mainly against the major CF bacterial pathogens. A high antibacterial activity against Staphylococcus aureus, including methicillin-resistant strains of this species, was observed. Bio-guided fractionation and analysis of the active fractions by nuclear magnetic resonance or by high-performance liquid chromatography and high-resolution electrospray ionization-mass spectrometry allowed us to identify boydone A as responsible for this antibacterial activity. Together, these results suggest that this six-membered cyclic polyketide could be one of the virulence factors of the fungus. Genes involved in the synthesis of this secreted metabolite are currently being identified in order to confirm the role of this polyketide in pathogenesis. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The killer of Socrates: Coniine and Related Alkaloids in the Plant Kingdom.

PubMed

Hotti, Hannu; Rischer, Heiko

2017-11-14

Coniine, a polyketide-derived alkaloid, is poisonous to humans and animals. It is a nicotinic acetylcholine receptor antagonist, which leads to inhibition of the nervous system, eventually causing death by suffocation in mammals. Coniine's most famous victim is Socrates who was sentenced to death by poison chalice containing poison hemlock in 399 BC. In chemistry, coniine holds two historical records: It is the first alkaloid the chemical structure of which was established (in 1881), and that was chemically synthesized (in 1886). In plants, coniine and twelve closely related alkaloids are known from poison hemlock ( Conium maculatum L.), and several Sarracenia and Aloe species. Recent work confirmed its biosynthetic polyketide origin. Biosynthesis commences by carbon backbone formation from butyryl-CoA and two malonyl-CoA building blocks catalyzed by polyketide synthase. A transamination reaction incorporates nitrogen from l-alanine and non-enzymatic cyclization leads to γ-coniceine, the first hemlock alkaloid in the pathway. Ultimately, reduction of γ-coniceine to coniine is facilitated by NADPH-dependent γ-coniceine reductase. Although coniine is notorious for its toxicity, there is no consensus on its ecological roles, especially in the carnivorous pitcher plants where it occurs. Lately there has been renewed interest in coniine's medical uses particularly for pain relief without an addictive side effect.

Redundant synthesis of a conidial polyketide by two distinct secondary metabolite clusters in Aspergillus fumigatus

PubMed Central

Throckmorton, Kurt; Lim, Fang Yun; Kontoyiannis, Dimitrios P.; Zheng, Weifa; Keller, Nancy P.

2016-01-01

Summary Filamentous fungi are renowned for the production of bioactive secondary metabolites. Typically, one distinct metabolite is generated from a specific secondary metabolite cluster. Here, we characterize the newly described trypacidin (tpc) cluster in the opportunistic human pathogen Aspergillus fumigatus. We find that this cluster as well as the previously characterized endocrocin (enc) cluster both contribute to the production of the spore metabolite endocrocin. Whereas trypacidin is eliminated when only tpc cluster genes are deleted, endocrocin production is only eliminated when both the tpc and enc non-reducing polyketide synthase-encoding genes, tpcC and encA, respectively, are deleted. EncC, an anthrone oxidase, converts the product released from EncA to endocrocin as a final product. In contrast, endocrocin synthesis by the tpc cluster likely results from incomplete catalysis by TpcK (a putative decarboxylase), as its deletion results in a nearly 10-fold increase in endocrocin production. We suggest endocrocin is likely a shunt product in all related non-reducing polyketide synthase clusters containing homologues of TpcK and TpcL (a putative anthrone oxidase), e.g. geodin and monodictyphenone. This finding represents an unusual example of two physically discrete secondary metabolite clusters generating the same natural product in one fungal species by distinct routes. PMID:26242966

ClusterCAD: a computational platform for type I modular polyketide synthase design

DOE PAGES

Eng, Clara H.; Backman, Tyler W H; Bailey, Constance B.; ...

2017-10-11

Here, we present ClusterCAD, a web-based toolkit designed to leverage the collinear structure and deterministic logic of type I modular polyketide synthases (PKSs) for synthetic biology applications. The unique organization of these megasynthases, combined with the diversity of their catalytic domain building blocks, has fueled an interest in harnessing the biosynthetic potential of PKSs for the microbial production of both novel natural product analogs and industrially relevant small molecules. However, a limited theoretical understanding of the determinants of PKS fold and function poses a substantial barrier to the design of active variants, and identifying strategies to reliably construct functional PKSmore » chimeras remains an active area of research. In this work, we formalize a paradigm for the design of PKS chimeras and introduce ClusterCAD as a computational platform to streamline and simplify the process of designing experiments to test strategies for engineering PKS variants. ClusterCAD provides chemical structures with stereochemistry for the intermediates generated by each PKS module, as well as sequence- and structure-based search tools that allow users to identify modules based either on amino acid sequence or on the chemical structure of the cognate polyketide intermediate. ClusterCAD can be accessed at https://clustercad.jbei.org and at http://clustercad.igb.uci.edu.« less

Phthalocyanine polymers

NASA Technical Reports Server (NTRS)

Achar, B. N.; Fohlen, G. M.; Parker, J. A. (Inventor)

1985-01-01

A method of forming 4,4',4'',4''' -tetraamino phthalocyanines involves reducing 4,4',4'',4''' -tetranitro phthalocyanines, polymerizing the metal tetraamino phthalocyanines with a tetracarboxylic dianhydride (preferably aromatic) or copolymerizing with a tetracarboxylic dianhydride and a diamine (preferably also aromatic) to produce amic acids which are then dehydrocyclized to imides. Thermally and oxidatively stable polymers result which form tough, flexible films, varnishes, adhesives, and fibers.

Method for producing heat-resistant semi-inorganic compounds

NASA Technical Reports Server (NTRS)

Yajima, S.; Okamura, K.; Shishido, T.; Hasegawa, Y.

1983-01-01

The method for producing a heat resistant, semi-inorganic compound is discussed. Five examples in which various alcohols, phenols, and aromatic carbonic acids are used to test heat resistance and solubility are provided.

Organic compounds in produced waters from coalbed natural gas wells in the Powder River Basin, Wyoming, USA

USGS Publications Warehouse

Orem, W.H.; Tatu, C.A.; Lerch, H.E.; Rice, C.A.; Bartos, T.T.; Bates, A.L.; Tewalt, S.; Corum, M.D.

2007-01-01

The organic composition of produced water samples from coalbed natural gas (CBNG) wells in the Powder River Basin, WY, sampled in 2001 and 2002 are reported as part of a larger study of the potential health and environmental effects of organic compounds derived from coal. The quality of CBNG produced waters is a potential environmental concern and disposal problem for CBNG producers, and no previous studies of organic compounds in CBNG produced water have been published. Organic compounds identified in the produced water samples included: phenols, biphenyls, N-, O-, and S-containing heterocyclic compounds, polycyclic aromatic hydrocarbons (PAHs), aromatic amines, various non-aromatic compounds, and phthalates. Many of the identified organic compounds (phenols, heterocyclic compounds, PAHs) are probably coal-derived. PAHs represented the group of organic compounds most commonly observed. Concentrations of total PAHs ranged up to 23 ??g/L. Concentrations of individual compounds ranged from about 18 to <0.01 ??g/L. Temporal variability of organic compound concentrations was documented, as two wells with relatively high organic compound contents in produced water in 2001 had much lower concentrations in 2002. In many areas, including the PRB, coal strata provide aquifers for drinking water wells. Organic compounds observed in produced water are also likely present in drinking water supplied from wells in the coal. Some of the organic compounds identified in the produced water samples are potentially toxic, but at the levels measured in these samples are unlikely to have acute health effects. The human health effects of low-level, chronic exposure to coal-derived organic compounds in drinking water are currently unknown. Continuing studies will evaluate possible toxic effects from low level, chronic exposure to coal-derived organic compounds in drinking water supplies.

Chemical, Physical and Biological Approaches to Prevent Ochratoxin Induced Toxicoses in Humans and Animals

PubMed Central

Varga, János; Kocsubé, Sándor; Péteri, Zsanett; Vágvölgyi, Csaba; Tóth, Beáta

2010-01-01

Ochratoxins are polyketide derived fungal secondary metabolites with nephrotoxic, immunosuppressive, teratogenic, and carcinogenic properties. Ochratoxin-producing fungi may contaminate agricultural products in the field (preharvest spoilage), during storage (postharvest spoilage), or during processing. Ochratoxin contamination of foods and feeds poses a serious health hazard to animals and humans. Several strategies have been investigated for lowering the ochratoxin content in agricultural products. These strategies can be classified into three main categories: prevention of ochratoxin contamination, decontamination or detoxification of foods contaminated with ochratoxins, and inhibition of the absorption of consumed ochratoxins in the gastrointestinal tract. This paper gives an overview of the strategies that are promising with regard to lowering the ochratoxin burden of animals and humans. PMID:22069658

Natural Products from the Lithistida: A Review of the Literature since 2000

PubMed Central

Winder, Priscilla L.; Pomponi, Shirley A.; Wright, Amy E.

2011-01-01

Lithistid sponges are known to produce a diverse array of compounds ranging from polyketides, cyclic and linear peptides, alkaloids, pigments, lipids, and sterols. A majority of these structurally complex compounds have very potent and interesting biological activities. It has been a decade since a thorough review has been published that summarizes the literature on the natural products reported from this amazing sponge order. This review provides an update on the current taxonomic classification of the Lithistida, describes structures and biological activities of 131 new natural products, and discusses highlights from the total syntheses of 16 compounds from marine sponges of the Order Lithistida providing a compilation of the literature since the last review published in 2002. PMID:22363244

Spectrophotometric Investigations of Macrolide Antibiotics: A Brief Review

PubMed Central

Keskar, Mrudul R; Jugade, Ravin M

2015-01-01

Macrolides, one of the most commonly used class of antibiotics, are a group of drugs produced by Streptomyces species. They belong to the polyketide class of natural products. Their activity is due to the presence of a large macrolide lactone ring with deoxy sugar moieties. They are protein synthesis inhibitors and broad-spectrum antibiotics, active against both gram-positive and gram-negative bacteria. Different analytical techniques have been reported for the determination of macrolides such as chromatographic methods, flow injection methods, spectrofluorometric methods, spectrophotometric methods, and capillary electrophoresis methods. Among these methods, spectrophotometric methods are sensitive and cost effective for the analysis of various antibiotics in pharmaceutical formulations as well as biological samples. This article reviews different spectrophotometric methods for the determination of macrolide antibiotics. PMID:26609215

Exploring flavour-producing core microbiota in multispecies solid-state fermentation of traditional Chinese vinegar

PubMed Central

Wang, Zong-Min; Lu, Zhen-Ming; Shi, Jin-Song; Xu, Zheng-Hong

2016-01-01

Multispecies solid-state fermentation (MSSF), a natural fermentation process driven by reproducible microbiota, is an important technique to produce traditional fermented foods. Flavours, skeleton of fermented foods, was mostly produced by microbiota in food ecosystem. However, the association between microbiota and flavours and flavour-producing core microbiota are still poorly understood. Here, acetic acid fermentation (AAF) of Zhenjiang aromatic vinegar was taken as a typical case of MSSF. The structural and functional dynamics of microbiota during AAF process was determined by metagenomics and favour analyses. The dominant bacteria and fungi were identified as Acetobacter, Lactobacillus, Aspergillus, and Alternaria, respectively. Total 88 flavours including 2 sugars, 9 organic acids, 18 amino acids, and 59 volatile flavours were detected during AAF process. O2PLS-based correlation analysis between microbiota succession and flavours dynamics showed bacteria made more contribution to flavour formation than fungi. Seven genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus and Staphylococcus were determined as functional core microbiota for production of flavours in Zhenjiang aromatic vinegar, based on their dominance and functionality in microbial community. This study provides a perspective for bridging the gap between the phenotype and genotype of ecological system, and advances our understanding of MSSF mechanisms in Zhenjiang aromatic vinegar. PMID:27241188

Exploring flavour-producing core microbiota in multispecies solid-state fermentation of traditional Chinese vinegar.

PubMed

Wang, Zong-Min; Lu, Zhen-Ming; Shi, Jin-Song; Xu, Zheng-Hong

2016-05-31

Multispecies solid-state fermentation (MSSF), a natural fermentation process driven by reproducible microbiota, is an important technique to produce traditional fermented foods. Flavours, skeleton of fermented foods, was mostly produced by microbiota in food ecosystem. However, the association between microbiota and flavours and flavour-producing core microbiota are still poorly understood. Here, acetic acid fermentation (AAF) of Zhenjiang aromatic vinegar was taken as a typical case of MSSF. The structural and functional dynamics of microbiota during AAF process was determined by metagenomics and favour analyses. The dominant bacteria and fungi were identified as Acetobacter, Lactobacillus, Aspergillus, and Alternaria, respectively. Total 88 flavours including 2 sugars, 9 organic acids, 18 amino acids, and 59 volatile flavours were detected during AAF process. O2PLS-based correlation analysis between microbiota succession and flavours dynamics showed bacteria made more contribution to flavour formation than fungi. Seven genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus and Staphylococcus were determined as functional core microbiota for production of flavours in Zhenjiang aromatic vinegar, based on their dominance and functionality in microbial community. This study provides a perspective for bridging the gap between the phenotype and genotype of ecological system, and advances our understanding of MSSF mechanisms in Zhenjiang aromatic vinegar.

Multiscale Evaluation of Catalytic Upgrading of Biomass Pyrolysis Vapors on Ni- and Ga-Modified ZSM-5

DOE PAGES

Yung, Matthew M.; Stanton, Alexander R.; Iisa, Kristiina; ...

2016-10-07

Metal-impregnated (Ni or Ga) ZSM-5 catalysts were studied for biomass pyrolysis vapor upgrading to produce hydrocarbons using three reactors constituting a 100 000x change in the amount of catalyst used in experiments. Catalysts were screened for pyrolysis vapor phase upgrading activity in two small-scale reactors: (i) a Pyroprobe with a 10 mg catalyst in a fixed bed and (ii) a fixed-bed reactor with 500 mg of catalyst. The best performing catalysts were then validated with a larger scale fluidized-bed reactor (using ~1 kg of catalyst) that produced measurable quantities of bio-oil for analysis and evaluation of mass balances. Despite somemore » inherent differences across the reactor systems (such as residence time, reactor type, analytical techniques, mode of catalyst and biomass feed) there was good agreement of reaction results for production of aromatic hydrocarbons, light gases, and coke deposition. Relative to ZSM-5, Ni or Ga addition to ZSM-5 increased production of fully deoxygenated aromatic hydrocarbons and light gases. In the fluidized bed reactor, Ga/ZSM-5 slightly enhanced carbon efficiency to condensed oil, which includes oxygenates in addition to aromatic hydrocarbons, and reduced oil oxygen content compared to ZSM-5. Ni/ZSM-5, while giving the highest yield of fully deoxygenated aromatic hydrocarbons, gave lower overall carbon efficiency to oil but with the lowest oxygen content. Reaction product analysis coupled with fresh and spent catalyst characterization indicated that the improved performance of Ni/ZSM-5 is related to decreasing deactivation by coking, which keeps the active acid sites accessible for the deoxygenation and aromatization reactions that produce fully deoxygenated aromatic hydrocarbons. The addition of Ga enhances the dehydrogenation activity of the catalyst, which leads to enhanced olefin formation and higher fully deoxygenated aromatic hydrocarbon yields compared to unmodified ZSM-5. Catalyst characterization by ammonia temperature programmed desorption, surface area measurements, and postreaction temperature-programmed oxidation (TPO) also showed that the metal-modified zeolites retained a greater percentage of their initial acidity and surface area, which was consistent between the reactor scales. These results demonstrate that the trends observed with smaller (milligram to gram) catalyst reactors are applicable to larger, more industrially relevant (kg) scales to help guide catalyst research toward application.« less

Investigating the Biosynthesis of Natural Products from Marine Proteobacteria: A Survey of Molecules and Strategies

PubMed Central

Timmermans, Marshall L.; Paudel, Yagya P.; Ross, Avena C.

2017-01-01

The phylum proteobacteria contains a wide array of Gram-negative marine bacteria. With recent advances in genomic sequencing, genome analysis, and analytical chemistry techniques, a whole host of information is being revealed about the primary and secondary metabolism of marine proteobacteria. This has led to the discovery of a growing number of medically relevant natural products, including novel leads for the treatment of multidrug-resistant Staphylococcus aureus (MRSA) and cancer. Of equal interest, marine proteobacteria produce natural products whose structure and biosynthetic mechanisms differ from those of their terrestrial and actinobacterial counterparts. Notable features of secondary metabolites produced by marine proteobacteria include halogenation, sulfur-containing heterocycles, non-ribosomal peptides, and polyketides with unusual biosynthetic logic. As advances are made in the technology associated with functional genomics, such as computational sequence analysis, targeted DNA manipulation, and heterologous expression, it has become easier to probe the mechanisms for natural product biosynthesis. This review will focus on genomics driven approaches to understanding the biosynthetic mechanisms for natural products produced by marine proteobacteria. PMID:28762997

Investigating the Biosynthesis of Natural Products from Marine Proteobacteria: A Survey of Molecules and Strategies.

PubMed

Timmermans, Marshall L; Paudel, Yagya P; Ross, Avena C

2017-08-01

The phylum proteobacteria contains a wide array of Gram-negative marine bacteria. With recent advances in genomic sequencing, genome analysis, and analytical chemistry techniques, a whole host of information is being revealed about the primary and secondary metabolism of marine proteobacteria. This has led to the discovery of a growing number of medically relevant natural products, including novel leads for the treatment of multidrug-resistant Staphylococcus aureus (MRSA) and cancer. Of equal interest, marine proteobacteria produce natural products whose structure and biosynthetic mechanisms differ from those of their terrestrial and actinobacterial counterparts. Notable features of secondary metabolites produced by marine proteobacteria include halogenation, sulfur-containing heterocycles, non-ribosomal peptides, and polyketides with unusual biosynthetic logic. As advances are made in the technology associated with functional genomics, such as computational sequence analysis, targeted DNA manipulation, and heterologous expression, it has become easier to probe the mechanisms for natural product biosynthesis. This review will focus on genomics driven approaches to understanding the biosynthetic mechanisms for natural products produced by marine proteobacteria.

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

PubMed Central

Fischbach, Michael A.; Lai, Jonathan R.; Roche, Eric D.; Walsh, Christopher T.; Liu, David R.

2007-01-01

Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with ≈10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (103 to 104 clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts. PMID:17620609

Dissociation reactions of protonated anthracycline antibiotics following electrospray ionization-tandem mass spectrometry

NASA Astrophysics Data System (ADS)

Sleno, Lekha; Campagna-Slater, Valerie; Volmer, Dietrich A.

2006-09-01

Fragmentation pathways of doxorubicin, a common cancer therapy agent, and three closely related analogs (epirubicin, daunorubicin, idarubicin) were compared using electrospray ionization with tandem mass spectrometry. This class of antibiotics with anti-tumour activity has important structural features, with a tetracyclic aromatic, polyketide portion, which is glycosylated with an amino sugar in order to exhibit its biological activity. Collision-induced dissociation spectra revealed very similar product ions for each analog, however, important differences were seen in the relative abundances and the ease at which certain fragments were formed. Fragment ions observed included those from cleavage of the glycosidic bond, loss of the side chain from the aglycone moiety, water losses and loss of a methyl radical. Following cleavage of the glycosidic bond, the charge can either reside on the aglycone portion or the sugar moiety, and each of these primary fragments undergoes several secondary dissociation pathways, depending on the collision energy. By ramping the collision voltage, we were able to correlate the changes in fragmentation behavior with small alterations in the structure of the precursor ion. The detailed study of the fragmentation behavior of doxorubicin was supported by accurate mass measurements, using an electrospray-time of flight instrument, as well as MS3 data from a quadrupole-linear ion trap mass spectrometer. Computational studies were also performed to help explain the role of certain functional groups in the fragmentation reactions.

Discriminating the reaction types of plant type III polyketide synthases

PubMed Central

Shimizu, Yugo; Ogata, Hiroyuki; Goto, Susumu

2017-01-01

Abstract Motivation: Functional prediction of paralogs is challenging in bioinformatics because of rapid functional diversification after gene duplication events combined with parallel acquisitions of similar functions by different paralogs. Plant type III polyketide synthases (PKSs), producing various secondary metabolites, represent a paralogous family that has undergone gene duplication and functional alteration. Currently, there is no computational method available for the functional prediction of type III PKSs. Results: We developed a plant type III PKS reaction predictor, pPAP, based on the recently proposed classification of type III PKSs. pPAP combines two kinds of similarity measures: one calculated by profile hidden Markov models (pHMMs) built from functionally and structurally important partial sequence regions, and the other based on mutual information between residue positions. pPAP targets PKSs acting on ring-type starter substrates, and classifies their functions into four reaction types. The pHMM approach discriminated two reaction types with high accuracy (97.5%, 39/40), but its accuracy decreased when discriminating three reaction types (87.8%, 43/49). When combined with a correlation-based approach, all 49 PKSs were correctly discriminated, and pPAP was still highly accurate (91.4%, 64/70) even after adding other reaction types. These results suggest pPAP, which is based on linear discriminant analyses of similarity measures, is effective for plant type III PKS function prediction. Availability and Implementation: pPAP is freely available at ftp://ftp.genome.jp/pub/tools/ppap/ Contact: goto@kuicr.kyoto-u.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:28334262

Bacterial genome mining of enzymatic tools for alkyne biosynthesis

PubMed Central

Zhu, Xuejun; Su, Michael; Manickam, Kadhirvel; Zhang, Wenjun

2015-01-01

The alkyne is an important functionality widely used in material science, pharmaceutical science, and chemical biology, but the importance of this functionality is contrasted by the very limited number of enzymes known to be involved in alkyne biosynthesis. We recently reported the first known carrier protein-dependent pathway for terminal alkyne formation, and in silico analysis suggested that this mechanism could be widespread in bacteria. In this paper, we screened additional homologous gene cassettes presumed to be involved in alkyne biosynthesis using both in vitro biochemical study and an E. coli-polyketide synthase (PKS) reporting system for in vivo analysis. We discovered and characterized a new terminal alkyne biosynthetic pathway comprised of TtuA, B, and C from Teredinibacter turnerae T7901. While the acyl-CoA ligase homolog (TtuA) demonstrated promiscuity in the activation and loading of medium-chain fatty acids onto the carrier protein (TtuC), the desaturase homolog (TtuB) showed stringent substrate specificity towards C10 fatty acyl moieties. In addition, TtuB was demonstrated to be a bifunctional desaturase/acetylenase that efficiently catalyzed two sequential O2-dependent dehydrogenation reactions. A novel terminal-alkyne bearing polyketide was further produced upon co-expression of ttuABC and a PKS gene in E. coli. The discovery and characterization of TtuA, B, and C provides us with a new bifunctional desaturase/acetylenase for mechanistic and structural study and expands the scarce enzyme inventory for the biosynthesis of the alkyne functionality, which has important applications in synthetic and chemical biology. PMID:26441143

Preclinical characterization of naturally occurring polyketide cyclophilin inhibitors from the sanglifehrin family.

PubMed

Gregory, Matthew A; Bobardt, Michael; Obeid, Susan; Chatterji, Udayan; Coates, Nigel J; Foster, Teresa; Gallay, Philippe; Leyssen, Pieter; Moss, Steven J; Neyts, Johan; Nur-e-Alam, Mohammad; Paeshuyse, Jan; Piraee, Mahmood; Suthar, Dipen; Warneck, Tony; Zhang, Ming-Qiang; Wilkinson, Barrie

2011-05-01

Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 μM and 0.16 μM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F<4%) and low solubility (<25 μM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2>5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.

Preclinical Characterization of Naturally Occurring Polyketide Cyclophilin Inhibitors from the Sanglifehrin Family▿†

PubMed Central

Gregory, Matthew A.; Bobardt, Michael; Obeid, Susan; Chatterji, Udayan; Coates, Nigel J.; Foster, Teresa; Gallay, Philippe; Leyssen, Pieter; Moss, Steven J.; Neyts, Johan; Nur-e-Alam, Mohammad; Paeshuyse, Jan; Piraee, Mahmood; Suthar, Dipen; Warneck, Tony; Zhang, Ming-Qiang; Wilkinson, Barrie

2011-01-01

Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 μM and 0.16 μM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F < 4%) and low solubility (<25 μM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2 > 5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties. PMID:21383094

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases.

PubMed

Lohman, Jeremy R; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

PubMed Central

Lohman, Jeremy R.; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

2015-01-01

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs. PMID:26420866

Antimicrobial potential and taxonomic investigation of piezotolerant Streptomyces sp. NIOT-Ch-40 isolated from deep-sea sediment.

PubMed

Padmanaban, Vishnu Priya; Verma, Pankaj; Venkatabaskaran, Srividhyalakshmi; Keppayan, Thirupathi; Gopal, Dharani; Sekar, Ashok Kumar; Ramalingam, Kirubagaran

2017-02-01

Microbial-derived natural products from extreme niches such as deepsea are known to possess structural and functional novelty. With this background, the present study was designed to investigate the bioprospecting potential and systematics of a deep-sea derived piezotolerant bacterial strain NIOT-Ch-40, showing affiliation to the genus Streptomyces based on 16S RNA gene similarity. Preliminary screening for the presence of biosynthetic genes like polyketide synthase I, polyketide synthase II, non ribosomal peptide synthase, 3-amino-5-hydroxybenzoic acid synthase and spiroindimicin followed by antibacterial activity testing confirmed the presence of potent bioactivity. The secondary metabolites produced during fermentation in Streptomyces broth at 28 °C for 7 days were extracted with ethyl acetate. The extract exhibited a specific inhibitory activity against Gram-positive bacteria and was significantly effective (p < 0.0001) against methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration and minimum bactericidal concentration against MRSA was 1.5 µg/mL, which was statistically significant in comparison with erythromycin. A multifaceted analysis of the Streptomyces spp. was carried out to delineate the strain NIOT-Ch-40 at a higher resolution which includes morphological, biochemical and molecular studies. Piezotolerance studies and comparison of fatty acid profiles at high pressures revealed that it could be considered as one of the taxonomic markers, especially for the strains isolated from the deep sea environments. In conclusion, the observation of comparative studies with reference strains indicated towards the strain NIOT-Ch-40 as an indigenous marine piezotolerant Streptomyces sp. with a higher probability of obtaining novel bioactive metabolites.

Mechanistic Insight into the Biosynthesis and Detoxification of Fumonisin Mycotoxins.

PubMed

Burgess, Kevin M N; Renaud, Justin B; McDowell, Tim; Sumarah, Mark W

2016-09-16

Fumonisins, notably FB1, FB2, FB3, and FB4, are economically important mycotoxins produced by a number Fusarium sp. that occur on corn, rice, and sorghum as well as by Aspergillus sp. on grapes. The fumonisin scaffold is comprised of a C18 polyketide backbone functionalized with two tricarballylic esters and an alanine derived amine. These functional groups contribute to fumonisin's ability to inhibit sphingolipid biosynthesis in animals, plants, and yeasts. We report for the first time the isolation and structure elucidation of two classes of nonaminated fumonisins (FPy and FLa) produced by Aspergillus welwitschiae. Using a Lemna minor (duckweed) bioassay, these new compounds were significantly less toxic in comparison to the fumonisin B mycotoxins, providing new insight into the mechanism of fumonisin toxicity. Time course fermentations monitoring the production of FB4, FPy4, and FLa4, as well as (13)C and (15)N stable isotope incorporation, suggest a novel postbiosynthetic oxidative deamination process for fumonisins. This pathway was further supported by a feeding study with FB1, a fumonisin not produced by Aspergillus sp., which resulted in its transformation to FPy1. This study demonstrates that Aspergillus have the ability to produce enzymes that could be used for fumonisin detoxification.

Stimulated anoxic biodegradation of aromatic hydrocarbons using Fe(III) ligands

USGS Publications Warehouse

Lovley, D.R.; Woodward, J.C.; Chapelle, F.H.

1994-01-01

Contamination of ground waters with water-soluble aromatic hydrocarbons, common components of petroleum pollution, often produces anoxic conditions under which microbial degradation of the aromatics is slow. Oxygen is often added to contaminated ground water to stimulate biodegradation, but this can be technically difficult and expensive. Insoluble Fe(III) oxides, which are generally abundant in shallow aquifers, are alternative potential oxidants, but are difficult for microorganisms to access. Here we report that adding organic ligands that bind to Fe(III) dramatically increases its bioavailability, and that in the presence of these ligands, rates of degradation of aromatic hydrocarbons in anoxic aquifer sediments are comparable to those in oxic sediments. We find that even benzene, which is notoriously refractory in the absence of oxygen, can be rapidly degraded. Our results suggest that increasing the bioavailability of Fe(III) by adding suitable ligands provides a potential alternative to oxygen addition for the bioremediation of petroleum-contaminated aquifers.Contamination of ground waters with water-soluble aromatic hydrocarbons, common components of petroleum pollution, often produces anoxic conditions under which microbial degradation of the aromatics is slow. Oxygen is often added to contaminated ground water to stimulate biodegradation, but this can be technically difficult and expensive. Insoluble Fe(III) oxides, which are generally abundant in shallow aquifers, are alternative potential oxidants, but are difficult for microorganisms to access. Here we report that adding organic ligands that bind to Fe(III) dramatically increases its bioavailability, and that in the presence of these ligands, rates of degradation of aromatic hydrocarbons in anoxic aquifer sediments are comparable to those in oxic sediments. We find that even benzene, which is notoriously refractory in the absence of oxygen, can be rapidly degraded. Our results suggest that increasing the bioavailability of Fe(III) by adding suitable ligands provides a potential alternative to oxygen addition for the bioremediation of petroleum-contamined aquifers.

Identification of PaPKS1, a polyketide synthase involved in melanin formation and its use as a genetic tool in Podospora anserina.

PubMed

Coppin, Evelyne; Silar, Philippe

2007-08-01

In the filamentous fungus Podospora anserina, many pigmentation mutations map to the median region of the complex locus '14', called segment '29'. The data presented in this paper show that segment 29 corresponds to a gene encoding a polyketide synthase, designated PaPKS1, and identifies two mutations that completely or partially abolish the activity of the PaPKS1 polypeptide. We present evidence that the P. anserina green pigment is a (DHN)-melanin. Using the powerful genetic system of PaPKS1 cloning, we demonstrate that in P. anserina trans-duplicated sequences are subject to the RIP process as previously demonstrated for the cis-duplicated regions.

Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

PubMed

Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

2009-03-04

The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

Genotype-driven isolation of enterocin with novel bioactivities from mangrove-derived Streptomyces qinglanensis 172205.

PubMed

Xu, Dong-Bo; Ma, Min; Deng, Zi-Xin; Hong, Kui

2015-07-01

The type II polyketide synthase (PKS) natural product enterocin (1) was isolated from a mangrove-derived novel species Streptomyces qinglanensis 172205 guided by genome sequence, and its putative biosynthetic gene cluster was revealed. Its natural analogues 5-deoxyenterocin (2) and wailupemycin A-C (3-5) were also identified by tandem mass spectrometry. By feeding experiments with aryl acids, strain 172205 was proved to incorporate partial exogenous starter units into enterocin- and wailupemycin-based analogues, thus being a new and suitable microorganism for engineering unnatural enc-derived polyketide metabolites. In addition, biological assays indicated that enterocin showed obvious inhibitory activity against β-amyloid protein (Aβ1-42) fibrillation and moderate cytotoxicity against HeLa and HepG2 for the first time.

Removal of crude oil polycyclic aromatic hydrocarbons via organoclay-microbe-oil interactions.

PubMed

Ugochukwu, Uzochukwu C; Fialips, Claire I

2017-05-01

Clay minerals are quite vital in biogeochemical processes but the effect of organo-clays in the microbial degradation of crude oil polycyclic aromatic hydrocarbons is not well understood. The role of organo-saponite and organo-montmorillonite in comparison with the unmodified clays in crude oil polycyclic aromatic hydrocarbons (PAHs) removal via adsorption and biodegradation was studied by carrying out microcosm experiments in aqueous clay/oil systems with a hydrocarbon degrading microbial community that is predominantly alcanivorax spp. Montmorillonite and saponite samples were treated with didecyldimethylammonium bromide to produce organo-montmorillonite and organo-saponite used in this study. Obtained results indicate that clays with high cation exchange capacity (CEC) such as montmorillonite produced organo-clay (organomontmorillonite) that was not stimulatory to biodegradation of crude oil polycyclic aromatic compounds, especially the low molecular weight (LMW) ones, such as dimethylnaphthalenes. It is suggested that interaction between the organic phase of the organo-clay and the crude oil PAHs which is hydrophobic in nature must have reduced the availability of the polycyclic aromatic hydrocarbons for biodegradation. Organo-saponite did not enhance the microbial degradation of dimethylnaphthalenes but enhanced the biodegradation of some other PAHs such as phenanthrene. The unmodified montmorillonite enhanced the microbial degradation of the PAHs and is most likely to have done so as a result of its high surface area that allows the accumulation of microbes and nutrients enhancing their contact. Copyright © 2017 Elsevier Ltd. All rights reserved.

Upgrading producer gas quality from rubber wood gasification in a radio frequency tar thermocatalytic treatment reactor.

PubMed

Anis, Samsudin; Zainal, Z A

2013-12-01

This study focused on improving the producer gas quality using radio frequency (RF) tar thermocatalytic treatment reactor. The producer gas containing tar, particles and water was directly passed at a particular flow rate into the RF reactor at various temperatures for catalytic and thermal treatments. Thermal treatment generates higher heating value of 5.76 MJ Nm(-3) at 1200°C. Catalytic treatments using both dolomite and Y-zeolite provide high tar and particles conversion efficiencies of about 97% on average. The result also showed that light poly-aromatic hydrocarbons especially naphthalene and aromatic compounds particularly benzene and toluene were still found even at higher reaction temperatures. Low energy intensive RF tar thermocatalytic treatment was found to be effective for upgrading the producer gas quality to meet the end user requirements and increasing its energy content. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bio-based production of monomers and polymers by metabolically engineered microorganisms.

PubMed

Chung, Hannah; Yang, Jung Eun; Ha, Ji Yeon; Chae, Tong Un; Shin, Jae Ho; Gustavsson, Martin; Lee, Sang Yup

2015-12-01

Recent metabolic engineering strategies for bio-based production of monomers and polymers are reviewed. In the case of monomers, we describe strategies for producing polyamide precursors, namely diamines (putrescine, cadaverine, 1,6-diaminohexane), dicarboxylic acids (succinic, glutaric, adipic, and sebacic acids), and ω-amino acids (γ-aminobutyric, 5-aminovaleric, and 6-aminocaproic acids). Also, strategies for producing diols (monoethylene glycol, 1,3-propanediol, and 1,4-butanediol) and hydroxy acids (3-hydroxypropionic and 4-hydroxybutyric acids) used for polyesters are reviewed. Furthermore, we review strategies for producing aromatic monomers, including styrene, p-hydroxystyrene, p-hydroxybenzoic acid, and phenol, and propose pathways to aromatic polyurethane precursors. Finally, in vivo production of polyhydroxyalkanoates and recombinant structural proteins having interesting applications are showcased. Copyright © 2015 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Settle, Amy E.; Berstis, Laura; Rorrer, Nicholas A.

In this tutorial review, we provide an overview of heterogeneous Diels–Alder catalysis for the production of lignocellulosic biomass-derived aromatic compounds. Diels–Alder reactions afford an extremely selective and efficient route for carbon–carbon cycloadditions to produce intermediates that can readily undergo subsequent dehydration or dehydrogenation reactions for aromatization. As a result, catalysis of Diels–Alder reactions with biomass-derived dienes and dienophiles has seen a growth of interest in recent years; however, significant opportunities remain to (i) tailor heterogeneous catalyst materials for tandem Diels–Alder and aromatization reactions, and (ii) utilize biomass-derived dienes and dienophiles to access both conventional and novel aromatic monomers. As such,more » this review discusses the mechanistic aspects of Diels–Alder reactions from both an experimental and computational perspective, as well as the synergy of Brønsted–Lewis acid catalysts to facilitate tandem Diels–Alder and aromatization reactions. Heterogeneous catalyst design strategies for Diels–Alder reactions are reviewed for two exemplary solid acid catalysts, zeolites and polyoxometalates, and recent efforts for targeting direct replacement aromatic monomers from biomass are summarized. In conclusion, we point out important research directions for progressing Diels–Alder catalysis to target novel, aromatic monomers with chemical functionality that enables new properties compared to monomers that are readily accessible from petroleum.« less

Strain Prioritization and Genome Mining for Enediyne Natural Products

PubMed Central

Yan, Xiaohui; Ge, Huiming; Huang, Tingting; Hindra; Yang, Dong; Teng, Qihui; Crnovčić, Ivana; Li, Xiuling; Rudolf, Jeffrey D.; Lohman, Jeremy R.; Gansemans, Yannick; Zhu, Xiangcheng; Huang, Yong; Zhao, Li-Xing; Jiang, Yi; Van Nieuwerburgh, Filip; Rader, Christoph

2016-01-01

ABSTRACT The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. PMID:27999165

CD1c presentation of synthetic glycolipid antigens with foreign alkyl branching motifs

PubMed Central

de Jong, Annemieke; Arce, Eva Casas; Cheng, Tan-Yun; van Summeren, Ruben P.; Feringa, Ben L.; Dudkin, Vadim; Crich, David; Matsunaga, Isamu; Minnaard, Adriaan J.; Moody, D. Branch

2009-01-01

Summary Human CD1c is a protein that activates αβ T cells by presenting self antigens, synthetic mannosyl phosphodolichols and mycobacterial mannosyl phosphopolyketides. To determine which molecular structures of antigens mediate a T cell response, we measured activation by structurally divergent M. tuberculosis mannosyl-β1-phosphomycoketides as well as by synthetic analogs produced by two methods that yield either stereorandom or stereospecific methyl branching patterns. T cell responses required both a phosphate and a β-linked mannose unit, and showed preference for C30–34 lipid units with methyl branches in the S-configuration. Thus, in all cases T cell responses were strongest for synthetic compounds that mimicked the natural branched lipids produced by mycobacterial polyketide synthase 12. Incorporation of methylmalonate to form branched lipids is a common bacterial lipid synthesis pathway that is absent in vertebrates, so the preferential recognition of branched lipids may represent a new type of lipid-based pathogen associated molecular pattern (PAMP). PMID:18022562

Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS–PKS hybrid enzyme

PubMed Central

Yun, Choong-Soo; Motoyama, Takayuki; Osada, Hiroyuki

2015-01-01

Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS–PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS–PKS hybrid enzyme. PMID:26503170

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen

2010-04-26

Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, themore » streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.« less

Chlorination of lignin by ubiquitous fungi has a likely role in global organochlorine production

PubMed Central

Ortiz-Bermúdez, Patricia; Hirth, Kolby C.; Srebotnik, Ewald; Hammel, Kenneth E.

2007-01-01

Soils and decayed plant litter contain significant quantities of chlorinated aromatic polymers that have a natural but largely unknown origin. We used cupric oxide ligninolysis coupled with gas chromatography/mass spectrometry to show that Curvularia inaequalis, a widely distributed litter ascomycete, chlorinated the aromatic rings of lignin in wood that it was degrading. In aspen wood decayed for 24 weeks, two chlorolignin fragments, 5-chlorovanillin and 2-chlorosyringaldehyde, were each found at ≈10 μg/g of wood (dry weight). These levels resemble those of similar structures generally found in unpolluted environmental samples. Fractionation of the extractable proteins followed by tandem mass spectrometric analysis showed that the colonized wood contained a previously described C. inaequalis chloroperoxidase that very likely catalyzed lignin chlorination. Chlorolignin produced by this route and humus derived from it are probably significant components of the global chlorine cycle because chloroperoxidase-producing fungi are ubiquitous in decaying lignocellulose and lignin is the earth's most abundant aromatic substance. PMID:17360449

o-Fluorination of aromatic azides yields improved azido-based fluorescent probes for hydrogen sulfide: synthesis, spectra, and bioimaging.

PubMed

Wei, Chao; Wang, Runyu; Wei, Lv; Cheng, Longhuai; Li, Zhifei; Xi, Zhen; Yi, Long

2014-12-01

Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule with multiple biological functions. To visualize the endogenous in situ production of H2S in real time, new coumarin- and boron-dipyrromethene-based fluorescent turn-on probes were developed for fast sensing of H2S in aqueous buffer and in living cells. Introduction of a fluoro group in the ortho position of the aromatic azide can lead to a greater than twofold increase in the rate of reaction with H2S. On the basis of o-fluorinated aromatic azides, fluorescent probes with high sensitivity and selectivity toward H2S over other biologically relevant species were designed and synthesized. The probes can be used to in situ to visualize exogenous H2S and D-cysteine-dependent endogenously produced H2S in living cells, which makes them promising tools for potential applications in H2S biology. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improvement of neutral oil quality in the production of sulfonate additives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhurba, A.S.; Bludilin, V.M.; Antonov, V.N.

This paper is concerned with improvement of neutral oil used as materials for sulfonation to produce additives for lubricating oils. In this article the authors analyze the basic reasons for the unsatisfactory quality of the neutral oil and attempt to define the ways in which the process technology can be improved so as to produce neutral oil with the required composition, at the same time raising the efficiency of utilization of the MSG-8 oil used as a feedstock for this process. Experimental results are presented which demonstrate the feasibility of sulfonating neutral oil in the high-speed mixer under near-optimal conditions.more » The yield of sulfonic acid approaches the theoretical yield. With the lowest contents of aromatic hydrocarbons in the original neutral oil, the aromatic hydrocarbons are almost completely converted to sulfonic acids. The yield of neutral oil is sufficiently high, and the residual content of aromatic hydrocarbons in the oil is no greater than 3%.« less

Renewable platform chemicals from directional microwave-assisted liquefaction coupling stepwise extraction of waste biomass.

PubMed

Feng, Junfeng; Hse, Chungyun; Yang, Zhongzhi; Wang, Kui; Jiang, Jianchun; Xu, Junming

2017-11-01

Directional microwave-assisted liquefaction and stepwise extraction are introduced for producing platform chemicals: aromatics and monosaccharides. When sulfuric acid was used as a catalyst, a 45% monosaccharides yield and a 29% aromatics yield were obtained from bamboo with 0.3g catalyst per 18g methanol and 2g bamboo at 160°C with 10min. Approximately 78-86wt% of the six biomass materials were converted into liquid products. After the stepwise extraction and precipitation process, the yields of monosaccharide derivatives and three phenolic compound fractions were 39-45% and 28-32%, respectively. Monosaccharides from holocellulose collected with a high purity of methyl glycosides were higher than 90%. Aromatic derivatives with different weight-molecular distributions were separated into three fractions with more than 80% phenolics. As their similar chemical properties within each fraction, platform chemicals have great commercial potential for producing high-quality chemicals and biofuels using mild upgrading conditions. Copyright © 2017. Published by Elsevier Ltd.

Solid-State NMR Investigation of Bio-chars Produced from Biomass Components and Whole Biomasses

DOE PAGES

Ben, Haoxi; Hao, Naijia; Liu, Qian; ...

2017-08-24

Bio-char is a by-product from thermochemical treatment of biomass and has been identified as an energy condensed product with a comparable heating value as commercial coal. However, the combustion of such solid product as an energy resource is only a preliminary application. It is highly possible to convert bio-char, which always has a condensed aromatic and porous structure to various high-value products. The investigations of the structures and formation pathways for the bio-char are very important to any future applications. In this study, six different biomass components, including cellulose, lignin, and tannin, and three whole biomasses—pine wood, pine residue, andmore » pine bark—have been used to produce bio-char at 400, 500, and 600 °C. Solid-state NMR and FT-IR have been employed in this study to characterize the structures for the bio-chars. The results indicated that the bio-chars produced from lignin contained some methoxyl groups, and the bio-chars produced from tannin contained significantly higher amount of phenolic hydroxyl groups. Compared to the bio-chars produced from pine wood and residue, the bio-chars produced from pine bark contained more aromatic C–O bonds, and aliphatic C–O and C–C bonds, which may be due to the significantly higher amount of lignin and tannin in the pine bark. Furthermore, the elevated amounts of aromatic C–O and aliphatic C–O and C–C bonds in the bio-chars from pine bark appeared to be completely decomposed at 600 °C.« less

Solid-State NMR Investigation of Bio-chars Produced from Biomass Components and Whole Biomasses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ben, Haoxi; Hao, Naijia; Liu, Qian

Bio-char is a by-product from thermochemical treatment of biomass and has been identified as an energy condensed product with a comparable heating value as commercial coal. However, the combustion of such solid product as an energy resource is only a preliminary application. It is highly possible to convert bio-char, which always has a condensed aromatic and porous structure to various high-value products. The investigations of the structures and formation pathways for the bio-char are very important to any future applications. In this study, six different biomass components, including cellulose, lignin, and tannin, and three whole biomasses—pine wood, pine residue, andmore » pine bark—have been used to produce bio-char at 400, 500, and 600 °C. Solid-state NMR and FT-IR have been employed in this study to characterize the structures for the bio-chars. The results indicated that the bio-chars produced from lignin contained some methoxyl groups, and the bio-chars produced from tannin contained significantly higher amount of phenolic hydroxyl groups. Compared to the bio-chars produced from pine wood and residue, the bio-chars produced from pine bark contained more aromatic C–O bonds, and aliphatic C–O and C–C bonds, which may be due to the significantly higher amount of lignin and tannin in the pine bark. Furthermore, the elevated amounts of aromatic C–O and aliphatic C–O and C–C bonds in the bio-chars from pine bark appeared to be completely decomposed at 600 °C.« less

Changing trends in biotechnology of secondary metabolism in medicinal and aromatic plants.

PubMed

Gandhi, Sumit G; Mahajan, Vidushi; Bedi, Yashbir S

2015-02-01

Medicinal and aromatic plants are known to produce secondary metabolites that find uses as flavoring agents, fragrances, insecticides, dyes and drugs. Biotechnology offers several choices through which secondary metabolism in medicinal plants can be altered in innovative ways, to overproduce phytochemicals of interest, to reduce the content of toxic compounds or even to produce novel chemicals. Detailed investigation of chromatin organization and microRNAs affecting biosynthesis of secondary metabolites as well as exploring cryptic biosynthetic clusters and synthetic biology options, may provide additional ways to harness this resource. Plant secondary metabolites are a fascinating class of phytochemicals exhibiting immense chemical diversity. Considerable enigma regarding their natural biological functions and the vast array of pharmacological activities, amongst other uses, make secondary metabolites interesting and important candidates for research. Here, we present an update on changing trends in the biotechnological approaches that are used to understand and exploit the secondary metabolism in medicinal and aromatic plants. Bioprocessing in the form of suspension culture, organ culture or transformed hairy roots has been successful in scaling up secondary metabolite production in many cases. Pathway elucidation and metabolic engineering have been useful to get enhanced yield of the metabolite of interest; or, for producing novel metabolites. Heterologous expression of putative plant secondary metabolite biosynthesis genes in a microbe is useful to validate their functions, and in some cases, also, to produce plant metabolites in microbes. Endophytes, the microbes that normally colonize plant tissues, may also produce the phytochemicals produced by the host plant. The review also provides perspectives on future research in the field.

Electrically conductive polyimides containing silver trifluoroacetylacetonate

NASA Technical Reports Server (NTRS)

Rancourt, James D. (Inventor); Stoakley, Diane M. (Inventor); Caplan, Maggie L. (Inventor); St. Clair, Anne K. (Inventor); Taylor, Larry T. (Inventor)

1996-01-01

Polyimides with enhanced electrical conductivity are produced by adding a silver ion-containing additive to the polyamic acid resin formed by the condensation of an aromatic dianhydride with an aromatic diamine. After thermal treatment the resulting polyimides had surface conductivities in the range of 1.7.times.10.sup.-3 4.5 .OMEGA..sup.-1 making them useful in low the electronics industry as flexible, electrically conductive polymeric films and coatings.

Renewable platform chemicals from directional microwave-assisted liquefaction coupling stepwise extraction of waste biomass

Treesearch

Junfeng Feng; Chungyun Hse; Zhongzhi Yang; Kui Wang; Jianchun Jiang; Junming Xu

2017-01-01

Directional microwave-assisted liquefaction and stepwise extraction are introduced for producing platform chemicals: aromatics and monosaccharides. When sulfuric acid was used as a catalyst, a 45% monosaccharides yield and a 29% aromatics yield were obtained from bamboo with 0.3 g catalyst per 18 g methanol and 2 g bamboo at 160 °C with 10 min. Approximately 78–86 wt%...

The evolution of floral scent and insect chemical communication.

PubMed

Schiestl, Florian P

2010-05-01

Plants have evolved a range of strategies to manipulate the behaviour of their insect partners. One powerful strategy is to produce signals that already have a role in the animals' own communication systems. To investigate to what extent the evolution of floral scents is correlated with chemical communication in insects, I analyse the occurrence, commonness, and evolutionary patterns of the 71 most common 'floral' volatile organic compounds (VOCs) in 96 plant families and 87 insect families. I found an overlap of 87% in VOCs produced by plants and insects. 'Floral' monoterpenes showed strong positive correlation in commonness between plants (both gymnosperms and angiosperms) and herbivores, whereas the commonness of 'floral' aromatics was positively correlated between angiosperms and both pollinators and herbivores. According to a multivariate regression analysis the commonness of 'floral' aromatics was best explained by their commonness in pollinators, whereas monoterpenes were best explained by herbivores. Among pollinator orders, aromatics were significantly more common in Lepidoptera than in Hymenoptera, whereas monoterpenes showed no difference among the two orders. Collectively, these patterns suggest that plants and insects converge in overall patterns of volatile production, both for attraction and defence. Monoterpenes seem to have evolved primarily for defence under selection by herbivores, whereas aromatics evolved signalling functions in angiosperms, primarily for pollinator attraction.

Phase I and phase II reductive metabolism simulation of nitro aromatic xenobiotics with electrochemistry coupled with high resolution mass spectrometry.

PubMed

Bussy, Ugo; Chung-Davidson, Yu-Wen; Li, Ke; Li, Weiming

2014-11-01

Electrochemistry combined with (liquid chromatography) high resolution mass spectrometry was used to simulate the general reductive metabolism of three biologically important nitro aromatic molecules: 3-trifluoromethyl-4-nitrophenol (TFM), niclosamide, and nilutamide. TFM is a pesticide used in the Laurential Great Lakes while niclosamide and nilutamide are used in cancer therapy. At first, a flow-through electrochemical cell was directly connected to a high resolution mass spectrometer to evaluate the ability of electrochemistry to produce the main reduction metabolites of nitro aromatic, nitroso, hydroxylamine, and amine functional groups. Electrochemical experiments were then carried out at a constant potential of -2.5 V before analysis of the reduction products by LC-HRMS, which confirmed the presence of the nitroso, hydroxylamine, and amine species as well as dimers. Dimer identification illustrates the reactivity of the nitroso species with amine and hydroxylamine species. To investigate xenobiotic metabolism, the reactivity of nitroso species to biomolecules was also examined. Binding of the nitroso metabolite to glutathione was demonstrated by the observation of adducts by LC-ESI(+)-HRMS and the characteristics of their MSMS fragmentation. In conclusion, electrochemistry produces the main reductive metabolites of nitro aromatics and supports the observation of nitroso reactivity through dimer or glutathione adduct formation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ijam, M.J.; Al-Qatami, S.Y.; Arif, S.F.

For several decades removal of aromatics from crude oil fractions has been practiced in oil refining to produce fuels and lubricants of lower aromatic content and hence of improved quality. These aromatics are suitable raw materials for the manufacture of aromatic solvents, aromatic process oils, high octane gasoline, and as basic materials for making detergents, perfumes and dyes. A study for the UV and IR spectra of the aromatic hydrocarbons showed them to consist mainly of bi-, tri-, tetra-, and penta-substituted benzene, bicyclic and tricyclic compounds. Detailed studies have been reported of molecular structure and substituent effects have been reportedmore » on the retention characteristics of aromatic hydrocarbons on alumina, silica and various chemically bonded silicas containing {minus}C{sub 18}, {minus}NH{sub 2}, {minus}R(NH){sub 2}, {minus}CN, RCN, and phenyl-mercuric acetate for compound class (ring-numbered) high performance liquid chromatography separation. With the aid of a Finnegan type 9612-4000 GC/MS apparatus, the mixture of neutral + basic aromatic hydrocarbons was qualitatively identified and revealed the presence of more than 112 peaks. The neutral + basic aromatic hydrocarbons consist mainly of: 3.68% monoaromatics (C{sub 3} - C{sub 6} alkyl benzenes), 52.81% bicycloaromatics (C{sub 0} - C{sub 4} alkylnaphthalenes), 6.20% tricycloaromatics (C{sub 0} - C{sub 4} alkyl phenanthrenes), and 37.32% nonhydrocarbons aromatic compounds. The components in major HPLC peaks corresponding to bicycloaromatics were further separated into small groups (3-4 components in each) by HPLC using an ODS-reverse phase-C{sub 18} column. To separate a single component from the mixture is a difficult problem. The individual compounds in the separated fractions were identified by GC/MS (Hewlett Packard 5993 system).« less

Effects of Alternative Fuels and Aromatics on Gas-Turbine Particle Emissions

NASA Astrophysics Data System (ADS)

Thornhill, K. L., II; Moore, R.; Winstead, E.; Anderson, B. E.; Klettlinger, J. L.; Ross, R. C.; Surgenor, A.

2015-12-01

This presentation describes experiments conducted with a Honeywell GTCP36-150 Auxiliary Power Unit (APU) to evaluate the effects of varying fuel composition on particle emissions. The APU uses a single-stage compressor stage, gas turbine engine with a can-type combustor to generate bypass flow and electrical power for supporting small aircraft and helicopters. It is installed in a "hush-house" at NASA Glenn Research Center and is configured as a stand-alone unit that can be fueled from an onboard tank or external supply. It operates at constant RPM, but its fuel flow can be varied by changing the electrical load or volume of bypass flow. For these tests, an external bank of resistors were attached to the APU's DC and AC electrical outlets and emissions measurements were made at low, medium and maximum electrical current loads. Exhaust samples were drawn from several points downstream in the exhaust duct and fed to an extensive suite of gas and aerosol sensors installed within a mobile laboratory parked nearby. Aromatic- and sulfur-free synthetic kerosenes from Rentech, Gevo, UOP, Amyris and Sasol were tested and their potential to reduce PM emissions evaluated against a single Jet A1 base fuel. The role of aromatic compounds in regulating soot emissions was also evaluated by adding metered amounts of aromatic blends (Aro-100, AF-Blend, SAK) and pure compounds (tetracontane and 1-methylnaphthalene) to a base alternative fuel (Sasol). Results show that, relative to Jet A1, alternative fuels reduce nonvolatile particle number emissions by 50-80% and--by virtue of producing much smaller particles—mass emissions by 65-90%; fuels with the highest hydrogen content produced the greatest reductions. Nonvolatile particle emissions varied in proportion to fuel aromatic content, with additives containing the most complex ring structures producing the greatest emission enhancements.

Mutagenicity of diesel exhaust particle extracts: influence of fuel composition in two diesel engines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clark, C.R.; Henderson, T.R.; Royer, R.E.

The influence of diesel fuel composition on mutagenicity of exhaust particle associated organic compounds has been investigated using nine fuels varying in aromatic content and distillation properties. The tests were conducted with Oldsmobile Delta-88 and Peugot 504 diesel cars operated according to the EPA Federal Test Procedure. The particulate exhaust from each test was collected on a filter, extracted in dichloromethane and the resulting extract evaluated for mutagenicity in Salmonella strain TA-100. Mutagenicity of extracts of particles collected from the Oldsmobile were highest in the higher aromatic content fuels (greater than 30%) but similar for intermediate (20%) and low (13%)more » aromatic content fuels. No influence of aromaticity on mutagenicity was observed in samples collected from the Peugeot under the same conditions. Thus, fuel aromatic content may enhance the production of mutagenic combustion products at higher concentrations, but may be dependent upon engine type. A good correlation was observed between mutagenicity of the particle extracts and the initial boiling point of the fuel (r . 0.89). Gas chromatography/mass spectrometric analysis of the aromatic fraction of the fuels showed that the fuel producing the most mutagenic combustion products was highest in phenanthrene type compounds.« less

Six New Polyketide Decalin Compounds from Mangrove Endophytic Fungus Penicillium aurantiogriseum 328#

PubMed Central

Ma, Yanhong; Li, Jing; Huang, Meixiang; Liu, Lan; Wang, Jun; Lin, Yongcheng

2015-01-01

Six new compounds with polyketide decalin ring, peaurantiogriseols A–F (1–6), along with two known compounds, aspermytin A (7), 1-propanone,3-hydroxy-1-(1,2,4a,5,6,7,8,8a-octahydro-2,5-dihydroxy-1,2,6-trimethyl-1-naphthalenyl) (8), were isolated from the fermentation products of mangrove endophytic fungus Penicillium aurantiogriseum 328#. Their structures were elucidated based on their structure analysis. The absolute configurations of compounds 1 and 2 were determined by 1H NMR analysis of their Mosher esters; the absolute configurations of 3–6 were determined by using theoretical calculations of electronic circular dichroism (ECD). Compounds 1–8 showed low inhibitory activity against human aldose reductase, no activity of inducing neurite outgrowth, nor antimicrobial activity. PMID:26473887

Multimodular biocatalysts for natural product assembly

NASA Astrophysics Data System (ADS)

Schwarzer, Dirk; Marahiel, Mohamed A.

2001-03-01

Nonribosomal peptides and polyketides represent a large class of natural products that show an extreme structural diversity and broad pharmacological relevance. They are synthesized from simple building blocks such as amino or carboxy acids and malonate derivatives on multimodular enzymes called nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), respectively. Although utilizing different substrates, NRPSs and PKSs show striking similarities in the modular architecture of their catalytic domains and product assembly-line mechanism. Among these compounds are well known antibiotics (penicillin, vancomycin and erythromycin) as well as potent immunosuppressive agents (cyclosporin, rapamycin and FK 506). This review focuses on the modular organization of NRPSs, PKSs and mixed NRPS/PKS systems and how modules and domains that build up the biosynthetic templates can be exploited for the rational design of recombinant enzymes capable of synthesizing novel compounds.

Selective Oxidation of Lignin Model Compounds.

PubMed

Gao, Ruili; Li, Yanding; Kim, Hoon; Mobley, Justin K; Ralph, John

2018-05-02

Lignin, the planet's most abundant renewable source of aromatic compounds, is difficult to degrade efficiently to welldefined aromatics. We developed a microwave-assisted catalytic Swern oxidation system using an easily prepared catalyst, MoO 2 Cl 2 (DMSO) 2 , and DMSO as the solvent and oxidant. It demonstrated high efficiency in transforming lignin model compounds containing the units and functional groups found in native lignins. The aromatic ring substituents strongly influenced the selectivity of β-ether phenolic dimer cleavage to generate sinapaldehyde and coniferaldehyde, monomers not usually produced by oxidative methods. Time-course studies on two key intermediates provided insight into the reaction pathway. Owing to the broad scope of this oxidation system and the insight gleaned with regard to its mechanism, this strategy could be adapted and applied in a general sense to the production of useful aromatic chemicals from phenolics and lignin. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fumonisin B(1)-nonproducing strains of Fusarium verticillioides cause maize (Zea mays) ear infection and ear rot.

PubMed

Desjardins, A E; Plattner, R D

2000-11-01

Fumonisins are polyketide mycotoxins produced by Fusarium verticillioides (synonym F. moniliforme), a major pathogen of maize (Zea mays) worldwide. Most field strains produce high levels of fumonisin B(1) (FB(1)) and low levels of the less-oxygenated homologues FB(2) and FB(3), but fumonisin B(1)-nonproducing field strains have been obtained by natural variation. To test the role of various fumonisins in pathogenesis on maize under field conditions, one strain producing FB(1), FB(2), and FB(3), one strain producing only FB(2), one strain producing only FB(3), and one fumonisin-nonproducing strain were applied to ears via the silk channel and on seeds at planting. Disease severity on the harvested ears was evaluated by visible symptoms and by weight percent symptomatic kernels. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of the applied FB(1)-nonproducing strains in kernels was determined by analysis of recovered strains for fumonisin production and other traits. All three FB(1)-nonproducing strains were able to infect ears following either silk-channel application or seed application at planting and were as effective as the FB(1)-producing strain in causing ear rot following silk-channel application. These results indicate that production of FB(1), FB(2), or FB(3) is not required for F. verticillioides to cause maize ear infection and ear rot.

Processing for maximizing the level of crystallinity in linear aromatic polyimides

NASA Technical Reports Server (NTRS)

St.clair, Terry L. (Inventor)

1991-01-01

The process of the present invention includes first treating a polyamide acid (such as LARC-TPI polyamide acid) in an amide-containing solvent (such as N-methyl pyrrolidone) with an aprotic organic base (such as triethylamine), followed by dehydrating with an organic dehydrating agent (such as acetic anhydride). The level of crystallinity in the linear aromatic polyimide so produced is maximized without any degradation in the molecular weight thereof.

Pd(II)/PhI(OAc)2 promoted direct cross coupling of glucals with aromatic acids.

PubMed

Begum, Zubeda; Shankar, G; Sirisha, K; Reddy, B V Subba

2018-05-22

A highly efficient oxidative C2-aroyloxylation of D-glucal with aromatic carboxylic acids has been achieved for the first time using 5 mol% Pd(OAc) 2 and 1 equiv of PhI(OAc) 2 to produce C2-aroyloxyglycals in good yields. The use of excess of PhI(OAc) 2 (2 equiv) provides C2-acyloxyglycal exclusively. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fuel property effects on Navy aircraft fuel systems

NASA Technical Reports Server (NTRS)

Moses, C. A.

1984-01-01

Problems of ensuring compatibility of Navy aircraft with fuels that may be different than the fuels for which the equipment was designed and qualified are discussed. To avoid expensive requalification of all the engines and airframe fuel systems, methodologies to qualify future fuels by using bench-scale and component testing are being sought. Fuel blends with increasing JP5-type aromatic concentration were seen to produce less volume swell than an equivalent aromatic concentration in the reference fuel. Futhermore, blends with naphthenes, decalin, tetralin, and naphthalenes do not deviate significantly from the correlation line of aromatic blends, Similar results are found with tensile strenth and elongation. Other elastomers, sealants, and adhesives are also being tested.

Infrared spectroscopy for the determination of hydrocarbon types in jet fuels

NASA Technical Reports Server (NTRS)

Buchar, C. S.

1981-01-01

The concentration of hydrocarbon types in conventional jet fuels and synfuels can be measured using a computerized infrared spectrophotometer. The computerized spectrophotometer is calibrated using a fuel of known aromatic and olefinic content. Once calibration is completed, other fuels can be rapidly analyzed using an analytical program built into the computer. The concentration of saturates can be calculated as 100 percent minus the sum of the aromatic and olefinic concentrations. The analysis of a number of jet fuels produced an average standard deviation of 1.76 percent for aromatic types and one of 3.99 percent for olefinic types. Other substances such as oils and organic mixtures can be analyzed for their hydrocarbon content.

Amination of electrophilic aromatic compounds by vicarious nucleophilic substitution

DOEpatents

Mitchell, Alexander R.; Pagoria, Philip F.; Schmidt, Robert D.

2000-01-01

The present invention relates to a process to aminate electrophilic aromatic compounds by vicarious nucleophilic substitution of hydrogen using quaternary hydrazinium salts. The use of trialkylhydrazinium halide, e.g., trimethylhydrazinium iodide, as well as hydroxylamine, alkoxylamines, and 4-amino-1,2,4-triazole to produce aminated aromatic structures, such as 1,3-diamino-2,4,6-trinitrobenzene (DATB), 1,3,5-triamino-2,4,6-trinitrobenzene (TATB) and 3,5-diamino-2,4,6-trinitrotoluene (DATNT), is described. DATB and TATB are useful insensitive high explosives. TATB is also used for the preparation of benzenehexamine, a starting material for the synthesis of novel materials (optical imaging devices, liquid crystals, ferromagnetic compounds).

The high-temperature oxidation of aromatic hydrocarbons

NASA Technical Reports Server (NTRS)

Brezinsky, K.

1986-01-01

Chemical mechanisms of the atmospheric pressure, high-temperature (875-1500 K) gas-phase oxidation of benzene, toluene, ethylbenzene, and propylbenzene are described and discussed. Oxidation trends evident from turbulent flow reactor experiments serve as the basis for the mechanisms of the oxidation of benzene and alkylated aromatics. The potential effects of very high temperatures and pressures on the chemistry of oxidation of aromatics are described. The oxidation of benzene and phenyl radical has been found to proceed in a stepwise C6-C5-C4 sequence. Species profiles obtained from flow-reactor experiments suggest that the oxidation of benzene and phenyl radical follows the generalized route via phenoxy, cyclopentadienyl and butadienyl radical. The oxidation of the C4 species branches into multiple pathways that yield copious amounts of ethylene and acetylene. Certain major trends are evident: the alkylated aromatics on initial attack either form styrene, benzyl radical or benzene. The styrene reacts further to produce a benzyl radical or benzene. The oxidation of an alkylated aromatic hydrocarbon appears eventually to reduce to the oxidation of either phenyl radical or benzene.

Aromatic Amino Acid-Derived Compounds Induce Morphological Changes and Modulate the Cell Growth of Wine Yeast Species

PubMed Central

González, Beatriz; Vázquez, Jennifer; Cullen, Paul J.; Mas, Albert; Beltran, Gemma; Torija, María-Jesús

2018-01-01

Yeasts secrete a large diversity of compounds during alcoholic fermentation, which affect growth rates and developmental processes, like filamentous growth. Several compounds are produced during aromatic amino acid metabolism, including aromatic alcohols, serotonin, melatonin, and tryptamine. We evaluated the effects of these compounds on growth parameters in 16 different wine yeasts, including non-Saccharomyces wine strains, for which the effects of these compounds have not been well-defined. Serotonin, tryptamine, and tryptophol negatively influenced yeast growth, whereas phenylethanol and tyrosol specifically affected non-Saccharomyces strains. The effects of the aromatic alcohols were observed at concentrations commonly found in wines, suggesting a possible role in microbial interaction during wine fermentation. Additionally, we demonstrated that aromatic alcohols and ethanol are able to affect invasive and pseudohyphal growth in a manner dependent on nutrient availability. Some of these compounds showed strain-specific effects. These findings add to the understanding of the fermentation process and illustrate the diversity of metabolic communication that may occur among related species during metabolic processes. PMID:29696002

In silico and experimental methods revealed highly diverse bacteria with quorum sensing and aromatics biodegradation systems--a potential broad application on bioremediation.

PubMed

Huang, Yili; Zeng, Yanhua; Yu, Zhiliang; Zhang, Jing; Feng, Hao; Lin, Xiuchun

2013-11-01

Phylogenetic overlaps between aromatics-degrading bacteria and acyl-homoserine-lactone (AHL) or autoinducer (AI) based quorum-sensing (QS) bacteria were evident in literatures; however, the diversity of bacteria with both activities had never been finely described. In-silico searching in NCBI genome database revealed that more than 11% of investigated population harbored both aromatic ring-hydroxylating-dioxygenase (RHD) gene and AHL/AI-synthetase gene. These bacteria were distributed in 10 orders, 15 families, 42 genus and 78 species. Horizontal transfers of both genes were common among them. Using enrichment and culture dependent method, 6 Sphingomonadales and 4 Rhizobiales with phenanthrene- or pyrene-degrading ability and AHL-production were isolated from marine, wetland and soil samples. Thin-layer-chromatography and gas-chromatography-mass-spectrum revealed that these Sphingomonads produced various AHL molecules. This is the first report of highly diverse bacteria that harbored both aromatics-degrading and QS systems. QS regulation may have broad impacts on aromatics biodegradation, and would be a new angle for developing bioremediation technology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Regulation of the Synthesis of the Angucyclinone Antibiotic Alpomycin in Streptomyces ambofaciens by the Autoregulator Receptor AlpZ and Its Specific Ligand▿

PubMed Central

Bunet, Robert; Mendes, Marta V.; Rouhier, Nicolas; Pang, Xiuhua; Hotel, Laurence; Leblond, Pierre; Aigle, Bertrand

2008-01-01

Streptomyces ambofaciens produces an orange pigment and the antibiotic alpomycin, both of which are products of a type II polyketide synthase gene cluster identified in each of the terminal inverted repeats of the linear chromosome. Five regulatory genes encoding Streptomyces antibiotic regulatory proteins (alpV, previously shown to be an essential activator gene; alpT; and alpU) and TetR family receptors (alpZ and alpW) were detected in this cluster. Here, we demonstrate that AlpZ, which shows high similarity to γ-butyrolactone receptors, is at the top of a pathway-specific regulatory hierarchy that prevents synthesis of the alp polyketide products. Deletion of the two copies of alpZ resulted in the precocious production of both alpomycin and the orange pigment, suggesting a repressor role for AlpZ. Consistent with this, expression of the five alp-located regulatory genes and of two representative biosynthetic structural genes (alpA and alpR) was induced earlier in the alpZ deletion strain. Furthermore, recombinant AlpZ was shown to bind to specific DNA sequences within the promoter regions of alpZ, alpV, and alpXW, suggesting direct transcriptional control of these genes by AlpZ. Analysis of solvent extracts of S. ambofaciens cultures identified the existence of a factor which induces precocious production of alpomycin and pigment in the wild-type strain and which can disrupt the binding of AlpZ to its DNA targets. This activity is reminiscent of γ-butyrolactone-type molecules. However, the AlpZ-interacting molecule(s) was shown to be resistant to an alkali treatment capable of inactivating γ-butyrolactones, suggesting that the AlpZ ligand(s) does not possess a lactone functional group. PMID:18296523

Structural basis for cyclization specificity of two Azotobacter type III polyketide synthases: a single amino acid substitution reverses their cyclization specificity.

PubMed

Satou, Ryutaro; Miyanaga, Akimasa; Ozawa, Hiroki; Funa, Nobutaka; Katsuyama, Yohei; Miyazono, Ken-ichi; Tanokura, Masaru; Ohnishi, Yasuo; Horinouchi, Sueharu

2013-11-22

Type III polyketide synthases (PKSs) show diverse cyclization specificity. We previously characterized two Azotobacter type III PKSs (ArsB and ArsC) with different cyclization specificity. ArsB and ArsC, which share a high sequence identity (71%), produce alkylresorcinols and alkylpyrones through aldol condensation and lactonization of the same polyketomethylene intermediate, respectively. Here we identified a key amino acid residue for the cyclization specificity of each enzyme by site-directed mutagenesis. Trp-281 of ArsB corresponded to Gly-284 of ArsC in the amino acid sequence alignment. The ArsB W281G mutant synthesized alkylpyrone but not alkylresorcinol. In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a small amount of alkylpyrone. These results indicate that this amino acid residue (Trp-281 of ArsB or Gly-284 of ArsC) should occupy a critical position for the cyclization specificity of each enzyme. We then determined crystal structures of the wild-type and G284W ArsC proteins at resolutions of 1.76 and 1.99 Å, respectively. Comparison of these two ArsC structures indicates that the G284W substitution brings a steric wall to the active site cavity, resulting in a significant reduction of the cavity volume. We postulate that the polyketomethylene intermediate can be folded to a suitable form for aldol condensation only in such a relatively narrow cavity of ArsC G284W (and presumably ArsB). This is the first report on the alteration of cyclization specificity from lactonization to aldol condensation for a type III PKS. The ArsC G284W structure is significant as it is the first reported structure of a microbial resorcinol synthase.

Regulation of the synthesis of the angucyclinone antibiotic alpomycin in Streptomyces ambofaciens by the autoregulator receptor AlpZ and its specific ligand.

PubMed

Bunet, Robert; Mendes, Marta V; Rouhier, Nicolas; Pang, Xiuhua; Hotel, Laurence; Leblond, Pierre; Aigle, Bertrand

2008-05-01

Streptomyces ambofaciens produces an orange pigment and the antibiotic alpomycin, both of which are products of a type II polyketide synthase gene cluster identified in each of the terminal inverted repeats of the linear chromosome. Five regulatory genes encoding Streptomyces antibiotic regulatory proteins (alpV, previously shown to be an essential activator gene; alpT; and alpU) and TetR family receptors (alpZ and alpW) were detected in this cluster. Here, we demonstrate that AlpZ, which shows high similarity to gamma-butyrolactone receptors, is at the top of a pathway-specific regulatory hierarchy that prevents synthesis of the alp polyketide products. Deletion of the two copies of alpZ resulted in the precocious production of both alpomycin and the orange pigment, suggesting a repressor role for AlpZ. Consistent with this, expression of the five alp-located regulatory genes and of two representative biosynthetic structural genes (alpA and alpR) was induced earlier in the alpZ deletion strain. Furthermore, recombinant AlpZ was shown to bind to specific DNA sequences within the promoter regions of alpZ, alpV, and alpXW, suggesting direct transcriptional control of these genes by AlpZ. Analysis of solvent extracts of S. ambofaciens cultures identified the existence of a factor which induces precocious production of alpomycin and pigment in the wild-type strain and which can disrupt the binding of AlpZ to its DNA targets. This activity is reminiscent of gamma-butyrolactone-type molecules. However, the AlpZ-interacting molecule(s) was shown to be resistant to an alkali treatment capable of inactivating gamma-butyrolactones, suggesting that the AlpZ ligand(s) does not possess a lactone functional group.

Biotechnology of polyketides: New breath of life for the novel antibiotic genetic pathways discovery through metagenomics

PubMed Central

Gomes, Elisângela Soares; Schuch, Viviane; de Macedo Lemos, Eliana Gertrudes

2013-01-01

The discovery of secondary metabolites produced by microorganisms (e.g., penicillin in 1928) and the beginning of their industrial application (1940) opened new doors to what has been the main medication source for the treatment of infectious diseases and tumors. In fact, approximately 80 years after the discovery of the first antibiotic compound, and despite all of the warnings about the failure of the “goose that laid the golden egg,” the potential of this wealth is still inexorable: simply adjust the focus from “micro” to “nano”, that means changing the look from microorganisms to nanograms of DNA. Then, the search for new drugs, driven by genetic engineering combined with metagenomic strategies, shows us a way to bypass the barriers imposed by methodologies limited to isolation and culturing. However, we are far from solving the problem of supplying new molecules that are effective against the plasticity of multi- or pan-drug-resistant pathogens. Although the first advances in genetic engineering date back to 1990, there is still a lack of high-throughput methods to speed up the screening of new genes and design new molecules by recombination of pathways. In addition, it is necessary an increase in the variety of heterologous hosts and improvements throughout the full drug discovery pipeline. Among numerous studies focused on this subject, those on polyketide antibiotics stand out for the large technical-scientific efforts that established novel solutions for the transfer/engineering of major metabolic pathways using transposons and other episomes, overcoming one of the main methodological constraints for the heterologous expression of major pathways. In silico prediction analysis of three-dimensional enzymatic structures and advances in sequencing technologies have expanded access to the metabolic potential of microorganisms. PMID:24688489

Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature

PubMed Central

Paolo, William F; Dadachova, Ekaterina; Mandal, Piyali; Casadevall, Arturo; Szaniszlo, Paul J; Nosanchuk, Joshua D

2006-01-01

Background Wangiella dermatitidis is a human pathogenic fungus that is an etiologic agent of phaeohyphomycosis. W. dermatitidis produces a black pigment that has been identified as a dihydroxynaphthalene melanin and the production of this pigment is associated with its virulence. Cell wall pigmentation in W. dermatitidis depends on the WdPKS1 gene, which encodes a polyketide synthase required for generating the key precursor for dihydroxynaphthalene melanin biosynthesis. Results We analyzed the effects of disrupting WdPKS1 on dihydroxynaphthalene melanin production and resistance to antifungal compounds. Transmission electron microscopy revealed that wdpks1Δ-1 yeast had thinner cell walls that lacked an electron-opaque layer compared to wild-type cells. However, digestion of the wdpks1Δ-1 yeast revealed small black particles that were consistent with a melanin-like compound, because they were acid-resistant, reacted with melanin-binding antibody, and demonstrated a free radical signature by electron spin resonance analysis. Despite lacking the WdPKS1 gene, the mutant yeast were capable of catalyzing the formation of melanin from L-3,4-dihyroxyphenylalanine. The wdpks1Δ-1 cells were significantly more susceptible to killing by voriconazole, amphotericin B, NP-1 [a microbicidal peptide], heat and cold, and lysing enzymes than the heavily melanized parental or complemented strains. Conclusion In summary, W. dermatitidis makes WdPKS-dependent and -independent melanins, and the WdPKS1-dependent deposition of melanin in the cell wall confers protection against antifungal agents and environmental stresses. The biological role of the WdPKS-independent melanin remains unclear. PMID:16784529

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohman, Jeremy; Ma, Ming; Osipiuk, Jerzy

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representationmore » of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.« less

Aromatic VOCs global influence in the ozone production

NASA Astrophysics Data System (ADS)

Cabrera-Perez, David; Pozzer, Andrea

2016-04-01

Aromatic hydrocarbons are a subgroup of Volatile Organic Compounds (VOCs) of special interest in the atmosphere of urban and semi-urban areas. Aromatics form a high fraction of VOCs, are highly reactive and upon oxidation they are an important source of ozone. These group of VOCs are released to the atmosphere by processes related to biomass burning and fossil fuel consumption, while they are removed from the atmosphere primarily by OH reaction and by dry deposition. In addition, a branch of aromatics (ortho-nitrophenols) produce HONO upon photolysis, which is responsible of certain amount of the OH recycling. Despite their importance in the atmosphere in anthropogenic polluted areas, the influence of aromatics in the ozone production remains largely unknown. This is of particular relevance, being ozone a pollutant with severe side effects on air quality, health and climate. In this work the atmospheric impacts at global scale of the most emitted aromatic VOCs in the gas phase (benzene, toluene, xylenes, ethylbenzene, styrene, phenol, benzaldehyde and trimethylbenzenes) are analysed and assessed. Specifically, the impact on ozone due to aromatic oxidation is estimated, as this is of great interest in large urban areas and can be helpful for developing air pollution control strategies. Further targets are the quantification of the NOx loss and the OH recycling due to aromatic oxidation. In order to investigate these processes, two simulations were performed with the numerical chemistry and climate simulation ECHAM/MESSy Atmospheric Chemistry (EMAC) model. The simulations compare two cases, one with ozone concentrations when aromatics are present or the second one when they are missing. Finally, model simulated ozone is compared against a global set of observations in order to better constrain the model accuracy.

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

PubMed

Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem clusters of pikromycin biosynthetic gene clusters. The 60 kb pikromycin biosynthetic gene cluster was isolated in a single integration pSBAC vector. Introduction of the pikromycin biosynthetic gene cluster into the pikromycin non-producing strains resulted in higher pikromycin production. The utility of the pSBAC system as a precise cloning tool for large-sized biosynthetic gene clusters was verified through heterologous expression of the pikromycin biosynthetic gene cluster. Moreover, this pSBAC-driven heterologous expression strategy was confirmed to be an ideal approach for production of low and inconsistent natural products such as pikromycin in S. venezuelae, implying that this strategy could be employed for development of a custom overexpression scheme of natural product biosynthetic gene clusters in actinomycetes.

Cyclic Lipopeptides of Bacillus amyloliquefaciens subsp. plantarum Colonizing the Lettuce Rhizosphere Enhance Plant Defense Responses Toward the Bottom Rot Pathogen Rhizoctonia solani.

PubMed

Chowdhury, Soumitra Paul; Uhl, Jenny; Grosch, Rita; Alquéres, Sylvia; Pittroff, Sabrina; Dietel, Kristin; Schmitt-Kopplin, Philippe; Borriss, Rainer; Hartmann, Anton

2015-09-01

The commercially available inoculant Bacillus amyloliquefaciens FZB42 is able to considerably reduce lettuce bottom rot caused by Rhizoctonia solani. To understand the interaction between FZB42 and R. solani in the rhizosphere of lettuce, we used an axenic system with lettuce bacterized with FZB42 and inoculated with R. solani. Confocal laser scanning microscopy showed that FZB42 could delay the initial establishment of R. solani on the plants. To show which secondary metabolites of FZB42 are produced under these in-situ conditions, we developed an ultra-high performance liquid chromatography coupled to time of flight mass spectrometry-based method and identified surfactin, fengycin, and bacillomycin D in the lettuce rhizosphere. We hypothesized that lipopeptides and polyketides play a role in enhancing the plant defense responses in addition to the direct antagonistic effect toward R. solani and used a quantitative real-time polymerase chain reaction-based assay for marker genes involved in defense signaling pathways in lettuce. A significant higher expression of PDF 1.2 observed in the bacterized plants in response to subsequent pathogen challenge showed that FZB42 could enhance the lettuce defense response toward the fungal pathogen. To identify if surfactin or other nonribosomally synthesized secondary metabolites could elicit the observed enhanced defense gene expression, we examined two mutants of FZB42 deficient in production of surfactin and the lipopetides and polyketides, by expression analysis and pot experiments. In the absence of surfactin and other nonribosomally synthesized secondary metabolites, there was no enhanced PDF 1.2-mediated response to the pathogen challenge. Pot experiment results showed that the mutants failed to reduce disease incidence in lettuce as compared with the FZB42 wild type, indicating, that surfactin as well as other nonribosomally synthesized secondary metabolites play a role in the actual disease suppression and on lettuce health. In conclusion, our study showed that nonribosomally synthesized secondary metabolites of FZB42 are actually produced in the lettuce rhizosphere and contribute to the disease suppression by mediating plant defense gene expression toward the pathogen R. solani.

Vitra-violet process for producing flame resistant polyamides and products produced thereby. [protective clothing for high oxygen environments

NASA Technical Reports Server (NTRS)

Toy, M. S.; Stringham, R. S. (Inventor)

1980-01-01

Aromatic polyamides with improved nonflammability characteristics are produced by contacting a polyamide substrate with a gaseous medium comprising a minor amount of a haloolefinic material and an inert diluent in the presence of light having sufficient energy to effect chemical addition of the haloolefin to the polyamide substrate.

SadA-Expressing Staphylococci in the Human Gut Show Increased Cell Adherence and Internalization.

PubMed

Luqman, Arif; Nega, Mulugeta; Nguyen, Minh-Thu; Ebner, Patrick; Götz, Friedrich

2018-01-09

A subgroup of biogenic amines, the so-called trace amines (TAs), are produced by mammals and bacteria and can act as neuromodulators. In the genus Staphylococcus, certain species are capable of producing TAs through the activity of staphylococcal aromatic amino acid decarboxylase (SadA). SadA decarboxylates aromatic amino acids to produce TAs, as well as dihydroxy phenylalanine and 5-hydroxytryptophan to thus produce the neurotransmitters dopamine and serotonin. SadA-expressing staphylococci were prevalent in the gut of most probands, where they are part of the human intestinal microflora. Furthermore, sadA-expressing staphylococci showed increased adherence to HT-29 cells and 2- to 3-fold increased internalization. Internalization and adherence was also increased in a sadA mutant in the presence of tryptamine. The α2-adrenergic receptor is required for enhanced adherence and internalization. Thus, staphylococci in the gut might contribute to gut activity and intestinal colonization. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Engineering cell factories for producing building block chemicals for bio-polymer synthesis.

PubMed

Tsuge, Yota; Kawaguchi, Hideo; Sasaki, Kengo; Kondo, Akihiko

2016-01-21

Synthetic polymers are widely used in daily life. Due to increasing environmental concerns related to global warming and the depletion of oil reserves, the development of microbial-based fermentation processes for the production of polymer building block chemicals from renewable resources is desirable to replace current petroleum-based methods. To this end, strains that efficiently produce the target chemicals at high yields and productivity are needed. Recent advances in metabolic engineering have enabled the biosynthesis of polymer compounds at high yield and productivities by governing the carbon flux towards the target chemicals. Using these methods, microbial strains have been engineered to produce monomer chemicals for replacing traditional petroleum-derived aliphatic polymers. These developments also raise the possibility of microbial production of aromatic chemicals for synthesizing high-performance polymers with desirable properties, such as ultraviolet absorbance, high thermal resistance, and mechanical strength. In the present review, we summarize recent progress in metabolic engineering approaches to optimize microbial strains for producing building blocks to synthesize aliphatic and high-performance aromatic polymers.

Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species.

PubMed

Zhao, Xin; Kuipers, Oscar P

2016-11-07

Gram-positive bacteria of the Bacillales are important producers of antimicrobial compounds that might be utilized for medical, food or agricultural applications. Thanks to the wide availability of whole genome sequence data and the development of specific genome mining tools, novel antimicrobial compounds, either ribosomally- or non-ribosomally produced, of various Bacillales species can be predicted and classified. Here, we provide a classification scheme of known and putative antimicrobial compounds in the specific context of Bacillales species. We identify and describe known and putative bacteriocins, non-ribosomally synthesized peptides (NRPs), polyketides (PKs) and other antimicrobials from 328 whole-genome sequenced strains of 57 species of Bacillales by using web based genome-mining prediction tools. We provide a classification scheme for these bacteriocins, update the findings of NRPs and PKs and investigate their characteristics and suitability for biocontrol by describing per class their genetic organization and structure. Moreover, we highlight the potential of several known and novel antimicrobials from various species of Bacillales. Our extended classification of antimicrobial compounds demonstrates that Bacillales provide a rich source of novel antimicrobials that can now readily be tapped experimentally, since many new gene clusters are identified.

Antifungal activity improved by coproduction of cyclodextrins and anabaenolysins in Cyanobacteria

PubMed Central

Shishido, Tania K.; Jokela, Jouni; Kolehmainen, Clara-Theresia; Fewer, David P.; Wahlsten, Matti; Wang, Hao; Rouhiainen, Leo; Rizzi, Ermanno; De Bellis, Gianluca; Permi, Perttu; Sivonen, Kaarina

2015-01-01

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 cholesterol-containing 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 α-, β-, and γ-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. PMID:26474830

Antimicrobial activity of pyrrocidines from Acremonium zeae against endophytes and pathogens of maize.

PubMed

Wicklow, Donald T; Poling, Stephen M

2009-01-01

Acremonium zeae produces pyrrocidines A and B, which are polyketide-amino acid-derived antibiotics, and is recognized as a seedborne protective endophyte of maize which augments host defenses against microbial pathogens causing seedling blights and stalk rots. Pyrrocidine A displayed significant in vitro activity against Aspergillus flavus and Fusarium verticillioides in assays performed using conidia as inoculum, with pyrrocidine A being more active than B. In equivalent assays performed with conidia or hyphal cells as inoculum, pyrrocidine A revealed potent activity against major stalk and ear rot pathogens of maize, including F. graminearum, Nigrospora oryzae, Stenocarpella (Diplodia) maydis, and Rhizoctonia zeae. Pyrrocidine A displayed significant activity against seed-rotting saprophytes A. flavus and Eupenicillium ochrosalmoneum, as well as seed-infecting colonists of the phylloplane Alternaria alternata, Cladosporium cladosporioides, and Curvularia lunata, which produces a damaging leaf spot disease. Protective endophytes, including mycoparasites which grow asymptomatically within healthy maize tissues, show little sensitivity to pyrrocidines. Pyrrocidine A also exhibited potent activity against Clavibacter michiganense subsp. nebraskense, causal agent of Goss's bacterial wilt of maize, and Bacillus mojaviense and Pseudomonas fluorescens, maize endophytes applied as biocontrol agents, but were ineffective against the wilt-producing bacterium Pantoea stewartii.

Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

PubMed Central

Averesch, Nils J. H.; Krömer, Jens O.

2018-01-01

The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Re)construction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations. PMID:29632862

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wear, Jr., John Edmund

The objective of this study was to examine the hypothesis that select functional groups of bacteria from pristine sites have an innate ability to degrade synthetic aromatics that often contaminate groundwater environments,due to exposure to naturally occurring recalcitrant aromatics in their environment. This study demonstrates that subsurface microbial communities are capable of utilizing lignin and humic acid breakdown products. Utilizers of these compounds were found to be present in most all the wells tested. Even the deepest aquifer tested had utilizers present for all six of the aromatics tested. Highest counts for the aromatics tested were observed with the naturallymore » occurring breakdown products of either lignin or humic acid. Carboxylic acids were found to be an important sole carbon source for groundwater bacteria possibly explained by the fact that they are produced by the oxidative cleavage of aromatic ring structures. The carbohydrate sole carbon sources that demonstrated the greatest densities were ones commonly associated with humics. This study indicates that utilization of naturally occurring aromatic compounds in the subsurface is an important nutritional source for groundwater bacteria. In addition, it suggests that adaptation to naturally occurring recalcitrant substrates is the origin of degradative pathways for xenobiotic compounds with analogous structure. This work has important implications for in situ bioremediation as a method of environmental cleanup.« less

Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop-mediated isothermal amplification (LAMP) reaction.

PubMed

Storari, M; von Rohr, R; Pertot, I; Gessler, C; Broggini, G A L

2013-04-01

To develop two assays based on the loop-mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes. Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction. The two LAMP assays permitted to quickly and specifically identify DNA from OTA-producing black aspergilli, as well as isolates grown in pure culture. Monitoring vineyards for the presence of OTA-producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA-producing black aspergilli. © 2013 The Society for Applied Microbiology.

A Synthetic Recursive “+1” Pathway for Carbon Chain Elongation

PubMed Central

Marcheschi, Ryan J.; Li, Han; Zhang, Kechun; Noey, Elizabeth L.; Kim, Seonah; Chaubey, Asha; Houk, K. N.; Liao, James C.

2013-01-01

Nature uses four methods of carbon chain elongation for the production of 2-ketoacids, fatty acids, polyketides, and isoprenoids. Using a combination of quantum mechanical (QM) modeling, protein–substrate modeling, and protein and metabolic engineering, we have engineered the enzymes involved in leucine biosynthesis for use as a synthetic “+1” recursive metabolic pathway to extend the carbon chain of 2-ketoacids. This modified pathway preferentially selects longer-chain substrates for catalysis, as compared to the non-recursive natural pathway, and can recursively catalyze five elongation cycles to synthesize bulk chemicals, such as 1-heptanol, 1-octanol, and phenylpropanol directly from glucose. The “+1” chemistry is a valuable metabolic tool in addition to the “+5” chemistry and “+2” chemistry for the biosynthesis of isoprenoids, fatty acids, or polyketides. PMID:22242720

Smenamides A and B, Chlorinated Peptide/Polyketide Hybrids Containing a Dolapyrrolidinone Unit from the Caribbean Sponge Smenospongia aurea. Evaluation of Their Role as Leads in Antitumor Drug Research

PubMed Central

Teta, Roberta; Irollo, Elena; Della Sala, Gerardo; Pirozzi, Giuseppe; Mangoni, Alfonso; Costantino, Valeria

2013-01-01

An in-depth study of the secondary metabolites contained in the Caribbean sponge Smenospongia aurea led to the isolation of smenamide A (1) and B (2), hybrid peptide/polyketide compounds containing a dolapyrrolidinone unit. Their structures were elucidated using high-resolution ESI-MS/MS and homo- and heteronuclear 2D NMR experiments. Structures of smenamides suggested that they are products of the cyanobacterial metabolism, and 16S rRNA metagenomic analysis detected Synechococcus spongiarum as the only cyanobacterium present in S. aurea. Smenamides showed potent cytotoxic activity at nanomolar levels on lung cancer Calu-1 cells, which for compound 1 is exerted through a clear pro-apoptotic mechanism. This makes smenamides promising leads for antitumor drug design. PMID:24217287

An automated Genomes-to-Natural Products platform (GNP) for the discovery of modular natural products.

PubMed

Johnston, Chad W; Skinnider, Michael A; Wyatt, Morgan A; Li, Xiang; Ranieri, Michael R M; Yang, Lian; Zechel, David L; Ma, Bin; Magarvey, Nathan A

2015-09-28

Bacterial natural products are a diverse and valuable group of small molecules, and genome sequencing indicates that the vast majority remain undiscovered. The prediction of natural product structures from biosynthetic assembly lines can facilitate their discovery, but highly automated, accurate, and integrated systems are required to mine the broad spectrum of sequenced bacterial genomes. Here we present a genome-guided natural products discovery tool to automatically predict, combinatorialize and identify polyketides and nonribosomal peptides from biosynthetic assembly lines using LC-MS/MS data of crude extracts in a high-throughput manner. We detail the directed identification and isolation of six genetically predicted polyketides and nonribosomal peptides using our Genome-to-Natural Products platform. This highly automated, user-friendly programme provides a means of realizing the potential of genetically encoded natural products.

Capturing Labile Sulfenamide and Sulfinamide Serum Albumin Adducts of Carcinogenic Arylamines by Chemical Oxidation

PubMed Central

Peng, Lijuan; Turesky, Robert J.

2013-01-01

Aromatic amines and heterocyclic aromatic amines (HAAs) are a class of structurally related carcinogens that are formed during the combustion of tobacco or during the high temperature cooking of meats. These procarcinogens undergo metabolic activation by N-oxidation of the exocyclic amine group to produce N-hydroxylated metabolites, which are critical intermediates implicated in toxicity and DNA damage. The arylhydroxylamines and their oxidized arylnitroso derivatives can also react with cysteine (Cys) residues of glutathione or proteins to form, respectively, sulfenamide and sulfinamide adducts. However, sulfur-nitrogen linked adducted proteins are often difficult to detect because they are unstable and undergo hydrolysis during proteolytic digestion. Synthetic N-oxidized intermediates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic HAA produced in cooked meats, and 4-aminobiphenyl, a carcinogenic aromatic amine present in tobacco smoke were reacted with human serum albumin (SA) and formed labile sulfenamide or sulfinamide adducts at the Cys34 residue. Oxidation of the carcinogen-modified SA with m-chloroperoxybenzoic acid (m-CPBA) produced the arylsulfonamide adducts, which were stable to heat and the chemical reduction conditions employed to denature SA. The sulfonamide adducts of PhIP and 4-ABP were identified, by liquid chromatography/mass spectrometry, in proteolytic digests of denatured SA. Thus, selective oxidation of arylamine-modified SA produces stable arylsulfonamide-SA adducts, which may serve as biomarkers of these tobacco and dietary carcinogens. PMID:23240913

Major contribution of the Ehrlich pathway for 2-phenylethanol/rose flavor production in Ashbya gossypii.

PubMed

Ravasio, Davide; Wendland, Jürgen; Walther, Andrea

2014-09-01

Aroma alcohols of fermented food and beverages are derived from fungal amino acids catabolism via the Ehrlich pathway. This linear pathway consists of three enzymatic reactions to form fusel alcohols. Regulation of some of the enzymes occurs on the transcriptional level via Aro80. The riboflavin overproducer Ashbya gossypii produces strong fruity flavours in contrast to its much less aromatic relative Eremothecium cymbalariae. Genome comparisons indicated that A. gossypii harbors genes for aromatic amino acid catabolism (ARO8a, ARO8b, ARO10, and ARO80) while E. cymbalariae only encodes ARO8a and thus lacks major components of aromatic amino acid catabolism. Volatile compound (VOC) analysis showed that both Eremothecium species produce large amounts of isoamyl alcohol while A. gossypii also produces high levels of 2-phenylethanol. Deletion of the A. gossypii ARO-genes did not confer any growth deficiencies. However, A. gossypii ARO-mutants (except Agaro8a) were strongly impaired in aroma production, particularly in the production of the rose flavour 2-phenylethanol. Conversely, overexpression of ARO80 via the AgTEF1 promoter resulted in 50% increase in VOC production. Together these data indicate that A. gossypii is a very potent flavour producer and that amongst the non-Saccharomyces biodiversity strains can be identified that could provide positive sensory properties to fermented beverages. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Assembly line termination in cylindrocyclophane biosynthesis: discovery of an editing type II thioesterase domain in a type I polyketide synthase† †Electronic supplementary information (ESI) available: Fig. S1–S12; Tables S1–S8, full experimental details and procedures, 1H and 13C NMR spectral data and HRMS data of compounds 10 and 11 and the internal standards. See DOI: 10.1039/c4sc03132f Click here for additional data file.

PubMed Central

Nakamura, H.; Wang, J. X.

2015-01-01

The termination step is an important source of structural diversity in polyketide biosynthesis. Most type I polyketide synthase (PKS) assembly lines are terminated by a thioesterase (TE) domain located at the C-terminus of the final module, while other PKS assembly lines lack a terminal TE domain and are instead terminated by a separate enzyme in trans. In cylindrocyclophane biosynthesis, the type I modular PKS assembly line is terminated by a freestanding type III PKS (CylI). Unexpectedly, the final module of the type I PKS (CylH) also possesses a C-terminal TE domain. Unlike typical type I PKSs, the CylH TE domain does not influence assembly line termination by CylI in vitro. Instead, this domain phylogenetically resembles a type II TE and possesses activity consistent with an editing function. This finding may shed light on the evolution of unusual PKS termination logic. In addition, the presence of related type II TE domains in many cryptic type I PKS and nonribosomal peptide synthetase (NRPS) assembly lines has implications for pathway annotation, product prediction, and engineering. PMID:29218151

Atlas of nonribosomal peptide and polyketide biosynthetic pathways reveals common occurrence of nonmodular enzymes.

PubMed

Wang, Hao; Fewer, David P; Holm, Liisa; Rouhiainen, Leo; Sivonen, Kaarina

2014-06-24

Nonribosomal peptides and polyketides are a diverse group of natural products with complex chemical structures and enormous pharmaceutical potential. They are synthesized on modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzyme complexes by a conserved thiotemplate mechanism. Here, we report the widespread occurrence of NRPS and PKS genetic machinery across the three domains of life with the discovery of 3,339 gene clusters from 991 organisms, by examining a total of 2,699 genomes. These gene clusters display extraordinarily diverse organizations, and a total of 1,147 hybrid NRPS/PKS clusters were found. Surprisingly, 10% of bacterial gene clusters lacked modular organization, and instead catalytic domains were mostly encoded as separate proteins. The finding of common occurrence of nonmodular NRPS differs substantially from the current classification. Sequence analysis indicates that the evolution of NRPS machineries was driven by a combination of common descent and horizontal gene transfer. We identified related siderophore NRPS gene clusters that encoded modular and nonmodular NRPS enzymes organized in a gradient. A higher frequency of the NRPS and PKS gene clusters was detected from bacteria compared with archaea or eukarya. They commonly occurred in the phyla of Proteobacteria, Actinobacteria, Firmicutes, and Cyanobacteria in bacteria and the phylum of Ascomycota in fungi. The majority of these NRPS and PKS gene clusters have unknown end products highlighting the power of genome mining in identifying novel genetic machinery for the biosynthesis of secondary metabolites.

A DFT study on photoinduced surface catalytic coupling reactions on nanostructured silver: selective formation of azobenzene derivatives from para-substituted nitrobenzene and aniline.

PubMed

Zhao, Liu-Bin; Huang, Yi-Fan; Liu, Xiu-Min; Anema, Jason R; Wu, De-Yin; Ren, Bin; Tian, Zhong-Qun

2012-10-05

We propose that aromatic nitro and amine compounds undergo photochemical reductive and oxidative coupling, respectively, to specifically produce azobenzene derivatives which exhibit characteristic Raman signals related to the azo group. A photoinduced charge transfer model is presented to explain the transformations observed in para-substituted ArNO(2) and ArNH(2) on nanostructured silver due to the surface plasmon resonance effect. Theoretical calculations show that the initial reaction takes place through excitation of an electron from the filled level of silver to the lowest unoccupied molecular orbital (LUMO) of an adsorbed ArNO(2) molecule, and from the highest occupied molecular orbital (HOMO) of an adsorbed ArNH(2) molecule to the unoccupied level of silver, during irradiation with visible light. The para-substituted ArNO(2)(-)˙ and ArNH(2)(+)˙ surface species react further to produce the azobenzene derivatives. Our results may provide a new strategy for the syntheses of aromatic azo dyes from aromatic nitro and amine compounds based on the use of nanostructured silver as a catalyst.

Produced water ponds are an important source of aromatics and alcohols in Rocky Mountain oil and gas basins

NASA Astrophysics Data System (ADS)

Lyman, S. N.

2017-12-01

Most of the water extracted with oil and natural gas (i.e., produced water) is disposed of by injection into the subsurface. In the arid western United States, however, a significant portion of produced water is discharged in ponds for evaporative disposal, and produced water is often stored in open ponds prior to subsurface injection. Even though they are common in the West (Utah's Uinta Basin has almost 200 ha), produced water ponds have been excluded from oil and gas emissions inventories because little information about their emission rates and speciation is available. We used flux chambers and inverse plume modeling to measure emissions of methane, C2-C11 hydrocarbons, light alcohols, carbonyls, and carbon dioxide from oil and gas produced water storage and disposal ponds in the Uinta Basin and the Upper Green River Basin, Wyoming, during 2013-2017. Methanol was the most abundant organic compound in produced water (91 ± 2% of the total volatile organic concentration; mean ± 95% confidence interval) but accounted for only 25 ± 30% of total organic compound emissions from produced water ponds. Non-methane hydrocarbons, especially C6-C9 alkanes and aromatics, accounted for the majority of emitted organics. We were able to predict emissions of individual compounds based on water concentrations, but only to within an order of magnitude. The speciation and magnitude of emissions varied strongly across facilities and was influenced by water age, the presence or absence of oil sheens, and with meteorological conditions (especially ice cover). Flux chamber measurements were lower than estimates from inverse modeling techniques.Based on our flux chamber measurements, we estimate that produced water ponds are responsible for between 3 and 9% of all non-methane organic compound emissions in the Uinta Basin (or as much as 18% if we rely on our inverse modeling results). Emissions from produced water ponds contain little methane and are more reactive (i.e., they have higher maximum incremental reactivity) than typical oil and gas-related emissions. Produced water ponds emit about 11% and 28%, respectively, of all aromatics and alcohols from the Uinta Basin oil and gas industry.

Process for converting light alkanes to higher hydrocarbons

DOEpatents

Noceti, Richard P.; Taylor, Charles E.

1988-01-01

A process is disclosed for the production of aromatic-rich, gasoline boiling range hydrocarbons from the lower alkanes, particularly from methane. The process is carried out in two stages. In the first, alkane is reacted with oxygen and hydrogen chloride over an oxyhydrochlorination catalyst such as copper chloride with minor proportions of potassium chloride and rare earth chloride. This produces an intermediate gaseous mixture containing water and chlorinated alkanes. The chlorinated alkanes are contacted with a crystalline aluminosilicate catalyst in the hydrogen or metal promoted form to produce gasoline range hydrocarbons with a high proportion of aromatics and a small percentage of light hydrocarbons (C.sub.2 -C.sub.4). The light hydrocarbons can be recycled for further processing over the oxyhydrochlorination catalyst.

Contaminated sediments from tributaries of the Great Lakes: Chemical characterization and carcinogenic effects in medaka (Oryzias latipes)

USGS Publications Warehouse

Besser, John M.; Schmitt, Christopher J.; Harshbarger, John C.; Peterman, Paul H.; Lebo, Jon A.

1991-01-01

Sediments from four inshore industrial sites and a reference site in the Great Lakes were extracted with organic solvents to produce a crude extract, which was separated on alumina into two fractions: predominantly polycyclic aromatic hydrocarbons; and predominantly nitrogencontaining polycyclic aromatic compounds. Crude extracts were redissolved in acetone and analyzed by gas chromatography and gas chromatography-mass spectrometry. The acetone-redissolved crude extracts from the four industrialized sites contained 5.6–313.3 μg total polycyclic aromatic compounds/g sediment and 3.0–36.4 μg other compounds/g sediment. In addition to the typical EPA priority pollutants, a substantial amount (228.7 μg/g sediment) of alkyl-polycyclic-aromatic compounds was detected in sediments from one of the industrialized sites. Extracts from the reference site contained 1.55 μg total polycyclic aromatic compounds/ g sediment. Medaka (Oryzias latipes) were exposed to multiple pulse doses of acetone-redissolved extracts and fractions. Medaka were also exposed to a known carcinogen, methylazoxymethanol acetate, to verify that chemicals produced tumors in the test fish. Acetone-redissolved extracts and fractions from contaminated sediments were toxic to medaka. Fin erosion and non-neoplastic liver abnormalities were more prevalent in medaka after exposure to acetoneredissolved extracts and fractions from contaminated sediments. Neoplasms previously associated with chemical exposure in wild fishes were induced in medaka exposed to acetone-redissolved extracts and fractions from two of the contaminated sites, but not from the reference site or controls. These findings further support the hypothesis that chemical contaminants in sediments are involved in epizootics of neoplasms in wild fishes at contaminated sites.

Alkylation of organic aromatic compounds

DOEpatents

Smith, L.A. Jr.

1989-07-18

Aromatic compounds are alkylated in a catalytic distillation, wherein the catalyst structure also serves as a distillation component by contacting the aromatic compound with a C[sub 2] to C[sub 10] olefin in the catalyst bed under 0.25 to 50 atmospheres of pressure and at temperatures in the range of 80 C to 500 C, using as the catalyst a mole sieve characterized as acidic or an acidic cation exchange resin. For example, ethyl benzene is produced by feeding ethylene below the catalyst bed while benzene is conveniently added through the reflux in molar excess to that required to react with ethylene, thereby reacting substantially all of the ethylene and recovering benzene as the principal overhead and ethyl benzene in the bottoms. 1 fig.

Alkylation of organic aromatic compounds

DOEpatents

Smith, Jr., Lawrence A.; Arganbright, Robert P.; Hearn, Dennis

1994-01-01

Aromatic compounds are alkylated in a catalytic distillation, wherein the catalyst structure also serves as a distillation component by contacting the aromatic compound with a C.sub.2 to C.sub.10 olefin in the catalyst bed under 0.25 to 50 atmospheres of pressure and at temperatures in the range of 80.degree. C. to 500.degree. C., using as the catalyst a mole sieve characterized as acidic or an acidic cation exchange resin. For example, ethyl benzene is produced by feeding ethylene below the catalyst bed while benzene is conveniently added through the reflux in molar excess to that required to react with ethylene, thereby reacting substantially all of the ethylene and recovering benzene as the principal overhead and ethyl benzene in the bottoms.

Alkylation of organic aromatic compounds

DOEpatents

Smith, Jr., Lawrence A.

1989-01-01

Aromatic compounds are alkylated in a catalytic distillation, wherein the catalyst structure also serves as a distillation component by contacting the aromatic compound with a C.sub.2 to C.sub.10 olefin in the catalyst bed under 0.25 to 50 atmospheres of pressure and at temperatures in the range of 80.degree. C. to 500.degree. C., using as the catalyst a mole sieve characterized as acidic or an acidic cation exchange resin. For example, ethyl benzene is produced by feeding ethylene below the catalyst bed while benzene is conveniently added through the reflux in molar excess to that required to react with ethylene, thereby reacting substantially all of the ethylene and recovering benzene as the principal overhead and ethyl benzene in the bottoms.

Alkylation of organic aromatic compounds

DOEpatents

Smith, L.A. Jr.; Arganbright, R.P.; Hearn, D.

1994-06-14

Aromatic compounds are alkylated in a catalytic distillation, wherein the catalyst structure also serves as a distillation component by contacting the aromatic compound with a C[sub 2] to C[sub 10] olefin in the catalyst bed under 0.25 to 50 atmospheres of pressure and at temperatures in the range of 80 C to 500 C, using as the catalyst a molecular sieve characterized as acidic or an acidic cation exchange resin. For example, ethyl benzene is produced by feeding ethylene below the catalyst bed while benzene is conveniently added through the reflux in molar excess to that required to react with ethylene, thereby reacting substantially all of the ethylene and recovering benzene as the principal overhead and ethyl benzene in the bottoms. 1 fig.

Evolution of Mycobacterium ulcerans and Other Mycolactone-Producing Mycobacteria from a Common Mycobacterium marinum Progenitor▿ †

PubMed Central

Yip, Marcus J.; Porter, Jessica L.; Fyfe, Janet A. M.; Lavender, Caroline J.; Portaels, Françoise; Rhodes, Martha; Kator, Howard; Colorni, Angelo; Jenkin, Grant A.; Stinear, Tim

2007-01-01

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans. PMID:17172337

Fighting mycobacterial infections by antibiotics, phytochemicals and vaccines.

PubMed

Bamberger, Denise; Jantzer, Nora; Leidner, Katharina; Arend, Joachim; Efferth, Thomas

2011-07-01

Buruli ulcer is a neglected disease caused by Mycobacterium ulcerans and represents the world's third most common mycobacterial infection. It produces the polyketide toxins, mycolactones A, B, C and D, which induce apoptosis and necrosis. Clinical symptoms are subcutaneous nodules, papules, plaques and ulcerating oedemae, which can enlarge and destroy nerves and blood vessels and even invade bones by lymphatic or haematogenous spread (osteomyelitis). Patients usually do not suffer from pain or systematic inflammation. Surgery is the treatment of choice, although recurrence is common and wide surgical excisions including healthy tissues result in significant morbidity. Antibiotic therapy with rifamycins, aminoglycosides, macrolides and quinolones also improves cure rates. Still less exploited treatment options are phytochemicals from medicinal plants used in affected countries. Vaccination against Buruli ulcer is still in its infancy. Copyright © 2010. Published by Elsevier SAS.

Mechanisms of fatty acid synthesis in marine fungus-like protists.

PubMed

Xie, Yunxuan; Wang, Guangyi

2015-10-01

Thraustochytrids are unicellular fungus-like protists and are well known for their ability to produce interesting nutraceutical compounds. Significant efforts have been made to improve their efficient production of important fatty acids (FAs), mostly by optimizing fermentation conditions and selecting highly productive thraustochytrid strains. Furthermore, noticeable improvements have been made in understanding the mechanism of FA biosynthesis, allowing for a better understanding of how thraustochytrids assemble these unique metabolites and how their biosynthesis is coupled with other related pathways. This review summarizes recent achievements on two major FA biosynthesis pathways, the standard pathway and the polyketide synthase pathway, and detail features of individual enzymes involved in FA biosynthesis, biotechnological advances in pathway engineering and enzyme characterization, and the discovery of other pathways that affect the efficiency of FA accumulation. Perspectives of biotechnological potential application of thraustochytrids are also discussed.

Involvement of secondary metabolites in the pathogenesis of the American foulbrood of honey bees caused by Paenibacillus larvae.

PubMed

Müller, Sebastian; Garcia-Gonzalez, Eva; Genersch, Elke; Süssmuth, Roderich D

2015-06-01

The Gram-positive, spore-forming bacterium Paenibacillus larvae (P. larvae) is the causative agent of the epizootic American Foulbrood (AFB), a fatal brood disease of the western honey bee (Apis mellifera). AFB is one of the most destructive honey bee diseases since it is not only lethal for infected larvae but also for the diseased colonies. Due to the high impact of honey bees on ecology and economy this epizootic is a severe and pressing problem. Knowledge about virulence mechanisms and the underlying molecular mechanisms remain largely elusive. Recent genome sequencing of P. larvae revealed its potential to produce unknown secondary metabolites, like nonribosomal peptides and peptide-polyketide hybrids. This article highlights recent findings on secondary metabolites synthesized by P. larvae and discusses their role in virulence and pathogenicity towards the bee larvae.

Reactor apparatus

DOEpatents

Echtler, J. Paul

1981-01-01

A reactor apparatus for hydrocracking a polynuclear aromatic hydrocarbonaceous feedstock to produce lighter hydrocarbon fuels by contacting the hydrocarbonaceous feedstock with hydrogen in the presence of a molten metal halide catalyst.

Generating Aromatics From CO2 on Mars or Natural Gas on Earth

NASA Technical Reports Server (NTRS)

Muscatello, Anthony C.; Zubrin, Robert; Berggren, Mark

2006-01-01

Methane to aromatics on Mars ( METAMARS ) is the name of a process originally intended as a means of converting Martian atmospheric carbon dioxide to aromatic hydrocarbons and oxygen, which would be used as propellants for spacecraft to return to Earth. The process has been demonstrated on Earth on a laboratory scale. A truncated version of the process could be used on Earth to convert natural gas to aromatic hydrocarbon liquids. The greater (relative to natural gas) density of aromatic hydrocarbon liquids makes it more economically feasible to ship them to distant markets. Hence, this process makes it feasible to exploit some reserves of natural gas that, heretofore, have been considered as being "stranded" too far from markets to be of economic value. In the full version of METAMARS, carbon dioxide is frozen out of the atmosphere and fed to a Sabatier reactor along with hydrogen (which, on Mars, would have been brought from Earth). In the Sabatier reactor, these feedstocks are converted to methane and water. The water is condensed and electrolyzed to oxygen (which is liquefied) and hydrogen (which is recycled to the Sabatier reactor). The methane is sent to an aromatization reactor, wherein, over a molybdenum-on-zeolite catalyst at a temperature 700 C, it is partially converted into aromatic hydrocarbons (specifically, benzene, toluene, and naphthalene) along with hydrogen. The aromatics are collected by freezing, while unreacted methane and hydrogen are separated by a membrane. Most of the hydrogen is recycled to the Sabatier reactor, while the methane and a small portion of the hydrogen are recycled to the aromatization reactor. The partial recycle of hydrogen to the aromatization reactor greatly increases the catalyst lifetime and eases its regeneration by preventing the formation of graphitic carbon, which could damage the catalyst. (Moreover, if graphitic carbon were allowed to form, it would be necessary to use oxygen to remove it.) Because the aromatics contain only one hydrogen atom per carbon atom, METAMARS produces four times as much propellant from a given amount of hydrogen as does a related process that includes the Sabatier reaction and electrolysis but not aromatization. In the terrestrial version of METAMARS, the Sabatier reactor and electrolyzer would be omitted, while the hydrogen/ methane membrane-separating membrane, the aromatization reactor, and the unreacted-gas-recycling subsystem would be retained. Natural gas would be fed directly to the aromatization reactor. Because natural gas consists of higher hydrocarbons in addition to methane, the aromatization subprocess should be more efficient than it is for methane alone.

Isomer effects on polyimide properties

NASA Technical Reports Server (NTRS)

Stump, B. L.

1978-01-01

Thermally stable polyimide polymers were prepared. Parameters explored include asymmetry of substitution, addition of alkyl substituents to an aromatic ring, and an increase in the number of aromatic rings present in the diamine monomer. It is shown that the use of an asymmetrical diamine in the preparation of a polyimide produces a polymer with a markedly lowered glass transition temperature. This is achieved with little or no sacrifice of thermal stability. An alternate approach taken was to prepare imide monomers which are capable of addition-type polymerization.

Fire and Pesticides: A Review of Air Quality Considerations

Treesearch

Parshall B. Bush; Daniel G. Neary; Charles K. McMahon

2000-01-01

The classes of primary chemical products naturally produced by the combustion of forest fuels are: carbon dioxide, water, carbon monoxide, particulate matter, methane and non-methane hydrocarbons, polynuclear aromatic hydrocarbons, nitrogen and sulfur oxides, aldehydes, free radicals, and inorganic elements. Secondary chemical products produced by reactions in smoke...

Emulsifier of Arthrobacter RAG-1: specificity of hydrocarbon substrate.

PubMed Central

Rosenberg, E; Perry, A; Gibson, D T; Gutnick, D L

1979-01-01

The purified extracellular emulsifying factor produced by Arthrobacter RAG-1 (EF-RAG) emulsified light petroleum oil, diesel oil, and a variety of crude oils and gas oils. Although kerosine and gasoline were emulsified poorly by EF-RAG, they were converted into good substrates for emulsification by addition of aromatic compounds, such as 2-methylnaphthalene. Neither aromatic nor aliphatic fractions of crude oil were emulsified by EF-RAG; however, mixtures containing both fractions were emulsified. Pure aliphatic or aromatic hydrocarbons were emulsified poorly by EF-RAG. Binary mixtures containing an aliphatic and an aromatic hydrocarbon, however, were excellent substrates for EF-RAG-induced emulsification. Of a variety of alkylcyclohexane and alkylbenzene derivatives tested, only hexyl- or heptylbenzene and octyl- or decylcyclohexane were effectively emulsified by EF-RAG. These data indicate that for EF-RAG to induce emulsification of hydrocarbons in water, the hydrocarbon substrate must contain both aliphatic and cyclic components. With binary mixtures of methylnaphthalene and hexadecane, maximum emulsion was obtained with 25% hexadecane. PMID:453821

Irradiated Benzene Ice Provides Clues to Meteoritic Organic Chemistry

NASA Technical Reports Server (NTRS)

Callahan, Michael Patrick; Gerakines, Perry Alexander; Martin, Mildred G.; Hudson, Reggie L.; Peeters, Zan

2013-01-01

Aromatic hydrocarbons account for a significant portion of the organic matter in carbonaceous chondrite meteorites, as a component of both the low molecular weight, solvent-extractable compounds and the insoluble organic macromolecular material. Previous work has suggested that the aromatic compounds in carbonaceous chondrites may have originated in the radiation-processed icy mantles of interstellar dust grains. Here we report new studies of the organic residue made from benzene irradiated at 19 K by 0.8 MeV protons. Polyphenyls with up to four rings were unambiguously identified in the residue by gas chromatography-mass spectrometry. Atmospheric pressure photoionization Fourier transform mass spectrometry was used to determine molecular composition, and accurate mass measurements suggested the presence of polyphenyls, partially hydrogenated polyphenyls, and other complex aromatic compounds. The profile of low molecular weight compounds in the residue compared well with extracts from the Murchison and Orgueil meteorites. These results are consistent with the possibility that solid phase radiation chemistry of benzene produced some of the complex aromatics found in meteorites.

Deuterium enrichment of polycyclic aromatic hydrocarbons by photochemically induced exchange with deuterium-rich cosmic ices

NASA Technical Reports Server (NTRS)

Sandford, S. A.; Bernstein, M. P.; Allamandola, L. J.; Gillette, J. S.; Zare, R. N.

2000-01-01

The polycyclic aromatic hydrocarbon (PAH) coronene (C24H12) frozen in D2O ice in a ratio of less than 1 part in 500 rapidly exchanges its hydrogen atoms with the deuterium in the ice at interstellar temperatures and pressures when exposed to ultraviolet radiation. Exchange occurs via three different chemical processes: D atom addition, D atom exchange at oxidized edge sites, and D atom exchange at aromatic edge sites. Observed exchange rates for coronene (C24H12)-D2O and d12-coronene (C24D12)-H2O isotopic substitution experiments show that PAHs in interstellar ices could easily attain the D/H levels observed in meteorites. These results may have important consequences for the abundance of deuterium observed in aromatic materials in the interstellar medium and in meteorites. These exchange mechanisms produce deuteration in characteristic molecular locations on the PAHs that may distinguish them from previously postulated processes for D enrichment of PAHs.

Mutagenicity of diesel exhaust particle extracts: influence of fuel composition in two diesel engines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clark, C.R.; Henderson, T.R.; Royer, R.E.

The influence of diesel fuel composition on mutagenicity of exhaust particle associated organic compounds has been investigated using nine fuels varying in aromatic content and distillation properties. The tests were conducted with Oldsmobile Delta-88 and Peugot 504 diesel cars operated according to the EPA Federal Test Procedure. The particulate exhaust from each test was collected on a filter, extracted in dichloromethane and the resulting extract evaluated for mutagenicity in Salmonella strain TA-100. Mutagenicity of extracts of particles collected from the Oldsmobile were highest in the higher aromatic content fuels (> 30%) but similar for intermediate (20%) and low (13%) aromaticmore » content fuels. No influence of aromaticity on mutagenicity was observed in samples collected from the Peugeot under the same conditions. Thus, fuel aromatic content may enhance the production of mutagenic combustion products at higher concentrations, but may be dependent upon engine type. A good correlation was observed between mutagenicity of the particle extracts and the initial boiling point of the fuel (r = 0.89). Gas chromatography/mass spectrometric analysis of the aromatic fraction of the fuels showed that the fuel producing the most mutagenic combustion products was highest in phenanthrene type compounds.« less

Ochratoxin A production by Penicillium thymicola.

PubMed

Nguyen, Hai D T; McMullin, David R; Ponomareva, Ekaterina; Riley, Robert; Pomraning, Kyle R; Baker, Scott E; Seifert, Keith A

2016-08-01

Ochratoxin A (OTA) is a mycotoxin produced by some Aspergillus and Penicillium species that grow on economically important agricultural crops and food products. OTA is classified as Group 2B carcinogen and is potently nephrotoxic, which is the basis for its regulation in some jurisdictions. Using high resolution mass spectroscopy, OTA and ochratoxin B (OTB) were detected in liquid culture extracts of Penicillium thymicola DAOMC 180753 isolated from Canadian cheddar cheese. The genome of this strain was sequenced, assembled and annotated to probe for putative genes involved in OTA biosynthesis. Known OTA biosynthetic genes from Penicillium verrucosum or Penicillium nordicum, two related Penicillium species that produce OTA, were not found in P. thymicola. However, a gene cluster containing a polyketide synthase (PKS) and PKS-nonribosomal peptide synthase (NRPS) hybrid encoding genes were located in the P. thymicola genome that showed a high degree of similarity to OTA biosynthetic enzymes of Aspergillus carbonarius and Aspergillus ochraceus. This is the first report of ochratoxin from P. thymicola and a new record of the species in Canada. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Unexpected Biotransformation of the HDAC Inhibitor Vorinostat Yields Aniline-Containing Fungal Metabolites.

PubMed

Adpressa, Donovon A; Stalheim, Kayla J; Proteau, Philip J; Loesgen, Sandra

2017-07-21

The diversity of genetically encoded small molecules produced by filamentous fungi remains largely unexplored, which makes these fungi an attractive source for the discovery of new compounds. However, accessing their full chemical repertoire under common laboratory culture conditions is a challenge. Epigenetic manipulation of gene expression has become a well-established tool for overcoming this obstacle. Here, we report that perturbation of the endophytic ascomycete Chalara sp. 6661, producer of the isofusidienol class of antibiotics, with the HDAC inhibitor vorinostat resulted in the production of four new modified xanthones. The structures of chalanilines A (1) and B (2) and adenosine-coupled xanthones A (3) and B (4) were determined by extensive NMR spectroscopic analyses, and the bioactivities of 1-4 were tested in antibiotic and cytotoxicity assays. Incorporation studies with deuterium-labeled vorinostat indicate that the aniline moiety in chalalanine A is derived from vorinostat itself. Our study shows that Chalara sp. is able to metabolize the HDAC inhibitor vorinostat to release aniline. This is a rare report of fungal biotransformation of the popular epigenetic modifier vorinostat into aniline-containing polyketides.

Metagenomic Analysis of the Sponge Discodermia Reveals the Production of the Cyanobacterial Natural Product Kasumigamide by 'Entotheonella'.

PubMed

Nakashima, Yu; Egami, Yoko; Kimura, Miki; Wakimoto, Toshiyuki; Abe, Ikuro

2016-01-01

Sponge metagenomes are a useful platform to mine cryptic biosynthetic gene clusters responsible for production of natural products involved in the sponge-microbe association. Since numerous sponge-derived bioactive metabolites are biosynthesized by the symbiotic bacteria, this strategy may concurrently reveal sponge-symbiont produced compounds. Accordingly, a metagenomic analysis of the Japanese marine sponge Discodermia calyx has resulted in the identification of a hybrid type I polyketide synthase-nonribosomal peptide synthetase gene (kas). Bioinformatic analysis of the gene product suggested its involvement in the biosynthesis of kasumigamide, a tetrapeptide originally isolated from freshwater free-living cyanobacterium Microcystis aeruginosa NIES-87. Subsequent investigation of the sponge metabolic profile revealed the presence of kasumigamide in the sponge extract. The kasumigamide producing bacterium was identified as an 'Entotheonella' sp. Moreover, an in silico analysis of kas gene homologs uncovered the presence of kas family genes in two additional bacteria from different phyla. The production of kasumigamide by distantly related multiple bacterial strains implicates horizontal gene transfer and raises the potential for a wider distribution across other bacterial groups.

Competitive interactions between sponge-associated bacteria.

PubMed

Esteves, Ana I S; Cullen, Alescia; Thomas, Torsten

2017-03-01

The diversity of the microbial communities associated with marine sponges has been extensively studied, but their functioning and interactions within the sponge holobiont are only recently being appreciated. Sponge-associated microorganisms are known for the production of a range of inhibitory metabolites with biotechnological application, but the ecological role that these compounds remains elusive. In this work, we explore the competitive interactions between cultivated sponge-associated bacteria to inspect whether bacteria that produce antimicrobial activities are able to inhibit potentially pathogenic bacteria. We isolated a Bacillus sp. bacterium with sponge-degrading activity, which likely has a negative impact on the host. This bacterium, along with other sponge isolates from the same genus, was found to be inhibited by a subpopulation of closely related sponge-derived Pseudovibrio spp. In some Pseudovibrio strains, these inhibitory activities were correlated with the genetic capacity to produce polyketides, such as erythronolide. Our observations suggest that antagonistic activities likely influence the composition of the sponge microbiome, including the abundance of bacteria that can be harmful to the host. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae

PubMed Central

Berry, J. P.; Reece, K. S.; Rein, K. S.; Baden, D. G.; Haas, L. W.; Ribeiro, W. L.; Shields, J. D.; Snyder, R. V.; Vogelbein, W. K.; Gawley, R. E.

2002-01-01

The estuarine genus Pfiesteria has received considerable attention since it was first identified and proposed to be the causative agent of fish kills along the mid-Atlantic coast in 1992. The presumption has been that the mechanism of fish death is by release of one or more toxins by the dinoflagellate. In this report, we challenge the notion that Pfiesteria species produce ichthyotoxins. Specifically, we show that (i) simple centrifugation, with and without ultrasonication, is sufficient to “detoxify” water of actively fish-killing cultures of Pfiesteria shumwayae, (ii) organic extracts of lyophilized cultures are not toxic to fish, (iii) degenerate primers that amplify PKS genes from several polyketide-producing dinoflagellates failed to yield a product with P. shumwayae DNA or cDNA, and (iv) degenerate primers for NRPS genes failed to amplify any NRPS genes but (unexpectedly) yielded a band (among several) that corresponded to known or putative PKSs and fatty acid synthases. We conclude that P. shumwayae is able to kill fish by means other than releasing a toxin into bulk water. Alternative explanations of the effects attributed to Pfiesteria are suggested. PMID:12163648

Observations of VOC emissions and photochemical products over US oil- and gas-producing regions using high-resolution H3O+ CIMS (PTR-ToF-MS)

NASA Astrophysics Data System (ADS)

Koss, Abigail; Yuan, Bin; Warneke, Carsten; Gilman, Jessica B.; Lerner, Brian M.; Veres, Patrick R.; Peischl, Jeff; Eilerman, Scott; Wild, Rob; Brown, Steven S.; Thompson, Chelsea R.; Ryerson, Thomas; Hanisco, Thomas; Wolfe, Glenn M.; St. Clair, Jason M.; Thayer, Mitchell; Keutsch, Frank N.; Murphy, Shane; de Gouw, Joost

2017-08-01

VOCs related to oil and gas extraction operations in the United States were measured by H3O+ chemical ionization time-of-flight mass spectrometry (H3O+ ToF-CIMS/PTR-ToF-MS) from aircraft during the Shale Oil and Natural Gas Nexus (SONGNEX) campaign in March-April 2015. This work presents an overview of major VOC species measured in nine oil- and gas-producing regions, and a more detailed analysis of H3O+ ToF-CIMS measurements in the Permian Basin within Texas and New Mexico. Mass spectra are dominated by small photochemically produced oxygenates and compounds typically found in crude oil: aromatics, cyclic alkanes, and alkanes. Mixing ratios of aromatics were frequently as high as those measured downwind of large urban areas. In the Permian, the H3O+ ToF-CIMS measured a number of underexplored or previously unreported species, including aromatic and cycloalkane oxidation products, nitrogen heterocycles including pyrrole (C4H5N) and pyrroline (C4H7N), H2S, and a diamondoid (adamantane) or unusual monoterpene. We additionally assess the specificity of a number of ion masses resulting from H3O+ ion chemistry previously reported in the literature, including several new or alternate interpretations.

Tagging polyketides/non-ribosomal peptides with a clickable functionality and applications

PubMed Central

Zhu, Xuejun; Zhang, Wenjun

2015-01-01

Bioorthogonal chemistry has recently emerged to be one of the most powerful tools in drug discovery and chemical biology. The exploration of it has successfully advanced the field of natural product research. In this Perspective, we survey current strategies for the installation of chemical handles into the molecular scaffolds of several major classes of natural products, including polyketides (PKs), non-ribosomal peptides (NRPs), and their hybrids. By tagging these natural products with chemical handles and coupling them with subsequent bioorthogonal reactions, researchers have visualized and studied the mode of action of natural products, as well as synthesized derivatives with better pharmaceutical properties. We conclude this Perspective by considering two questions: is there a general way to synthesize tagged PKs/NRPs? Does natural product labeling have a broader impact in the field of natural product research beyond current known applications? PMID:25815285

Polyketide derivatives from a marine-sponge-associated fungus Pestalotiopsis heterocornis.

PubMed

Lei, Hui; Lin, Xiuping; Han, Li; Ma, Jian; Dong, Kailin; Wang, Xingbo; Zhong, Jialiang; Mu, Yu; Liu, Yonghong; Huang, Xueshi

2017-10-01

Twelve previously undescribed polyketide derivatives, heterocornols A-L, and seven known analogues were isolated from a culture of the fungus Pestalotiopsis heterocornis associated with sponge. Their structures were elucidated by a comprehensive spectroscopic data analysis and CD Cotton effects. These compounds were evaluated for cytotoxic and antibacterial activities in vitro. Among them, heterocornols A-C, F-H, methyl-(2-formyl-3-hydroxyphenyl)propanoate, agropyrenol, and vaccinol G exhibited cytotoxicities against four human cancer cell lines with IC 50 values 15-100 μM, and they also showed antibacterial activities against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with MIC values ranging from 25 to 100 μg/mL. Moreover, compounds heterocornol C, heterocornol G, agropyrenol, and vaccinol G showed weak antifungal activities against Candida parapsilosis and Cryptococcus neoformans with MIC values 100 μg/mL. Copyright © 2017 Elsevier Ltd. All rights reserved.

Paenilamicin: structure and biosynthesis of a hybrid nonribosomal peptide/polyketide antibiotic from the bee pathogen Paenibacillus larvae.

PubMed

Müller, Sebastian; Garcia-Gonzalez, Eva; Mainz, Andi; Hertlein, Gillian; Heid, Nina C; Mösker, Eva; van den Elst, Hans; Overkleeft, Herman S; Genersch, Elke; Süssmuth, Roderich D

2014-09-26

The spore-forming bacterium Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a fatal disease of honey bees that occurs worldwide. Previously, we identified a complex hybrid nonribosomal peptide/polyketide synthesis (NRPS/PKS) gene cluster in the genome of P. larvae. Herein, we present the isolation and structure elucidation of the antibacterial and antifungal products of this gene cluster, termed paenilamicins. The unique structures of the paenilamicins give deep insight into the underlying complex hybrid NRPS/PKS biosynthetic machinery. Bee larval co-infection assays reveal that the paenilamicins are employed by P. larvae in fighting ecological niche competitors and are not directly involved in killing the bee larvae. Their antibacterial and antifungal activities qualify the paenilamicins as attractive candidates for drug development. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biosynthetic studies on the botcinolide skeleton: new hydroxylated lactones from Botrytis cinerea.

PubMed

Reino, José L; Durán-Patrón, Rosa M; Daoubi, Mourad; Collado, Isidro G; Hernández-Galán, Rosario

2006-01-20

[reaction: see text] The biosynthetic origin of the botcinolide skeleton was investigated by means of feeding 13C- and 2H-labeled precursors to Botrytis cinerea. Three new compounds, two homobotcinolide derivatives, 3-O-acetylhomobotcinolide (5) and 8-methylhomobotcinolide (6), and a new 11-membered lactone (7), were isolated. Their structures were elucidated on the basis of spectroscopic data, including one-bond and long-range 1H-13C correlations. The relative stereochemistries were determined by combined analyses of NOE data and 1H-1H coupling constants. According to the results of feeding experiments with 13C- and 2H-labeled acetate and l-S-methylmethionine, 5 is an acetate-derived polyketide whose methyl groups originate from l-S-methylmethionine. This is a rare example of the incorporation of a methyl from methionine into a supposed C3 starter unit of the polyketide synthesis.

Cloning and heterologous expression of genes from the kinamycin biosynthetic pathway of Streptomyces murayamaensis.

PubMed

Gould, S J; Hong, S T; Carney, J R

1998-01-01

The genes for most of the biosynthesis of the kinamycin antibiotics have been cloned and heterologously expressed. Genomic DNA of Streptomyces murayamaensis was partially digested with MboI and a library of approximately 40 kb fragments in E. coli XL1-BlueMR was prepared using the cosmid vector pOJ446. Hybridization with the actI probe from the actinorhodin polyketide synthase genes identified two clusters of polyketide genes. After transferal of these clusters to S. lividans ZX7, expression of one cluster was established by HPLC with photodiode array detection. Peaks were identified from the kin cluster for dehydrorabelomycin, kinobscurinone, and stealthin C, which are known intermediates in kinamycin biosynthesis. Two shunt metabolites, kinafluorenone and seongomycin were also identified. The structure of the latter was determined from a quantity obtained from large-scale fermentation of one of the clones.

Type II thioesterase gene (ECO-orf27) from Amycolatopsis orientalis influences production of the polyketide antibiotic, ECO-0501 (LW01).

PubMed

Shen, Yang; Huang, He; Zhu, Li; Luo, Minyu; Chen, Daijie

2012-11-01

ECO-orf27 associated with the cluster of ECO-0501 (LW01) from Amycolatopsis orientalis is deduced to encode a type II thioesterase. Disruption of ECO-orf27 reduced LW01 production by 95 %. Complementation of the disrupted mutant with intact ECO-orf27 restored the production of LW01 suggesting that ECO-orf27 is crucial for LW01 biosynthesis. ECO-TE I, the gene encoding type I thioesterase from LW01 polyketide synthases, cannot complement ECO-orf27 deficient mutant distinguishing ECO-orf27 from type I thioesterase gene. Type II thioesterase gene pikAV from Streptomyces venezuelae could complement ECO-orf27 in A. orientalis indicating that the two genes are equivalent in their function. Overexpression of ECO-orf27 resulted in a 20 % increase in LW01 production providing an alternative approach for yield improvement.

Determination of total and polycyclic aromatic hydrocarbons in aviation jet fuel.

PubMed

Bernabei, M; Reda, R; Galiero, R; Bocchinfuso, G

2003-01-24

The aviation jet fuel widely used in turbine engine aircraft is manufactured from straight-run kerosene. The combustion quality of jet fuel is largely related to the hydrocarbon composition of the fuel itself; paraffins have better burning properties than aromatic compounds, especially naphthalenes and light polycyclic aromatic hydrocarbons (PAHs), which are characterised as soot and smoke producers. For this reason the burning quality of fuel is generally measured as smoke fermation. This evaluation is carried out with UV spectrophotometric determination of total naphthalene hydrocarbons and a chromatographic analysis to determine the total aromatic compounds. These methods can be considered insufficient to evaluate the human health impact of these compounds due to their inability to measure trace (ppm) amounts of each aromatic hyrcarbon and each PAH in accordance with limitations imposed because of their toxicological properties. In this paper two analytical methods are presented. Both are based on a gas chromatographic technique with a mass detector operating in be selected ion monitoring mode. The first method was able to determine more than 60 aromatic hydrocarbons in a fuel sample in a 35-min chromatographic run, while the second was able to carry out the analysis of more than 30 PAHs in a 40-min chromatographic run. The linearity and sensitivity of the methods in measuring these analytes at trace levels are described.

Inhibition of hydroxyl radical reaction with aromatics by dissolved natural organic matter

USGS Publications Warehouse

Lindsey, M.E.; Tarr, M.A.

2000-01-01

Reaction of aromatic compounds with hydroxyl radical is inhibited by dissolved natural organic matter (NOM). The degree of inhibition is significantly greater than that expected based on a simple model in which aromatic compound molecules bound to NOM are considered to be unreactive. In this study, hydroxyl radical was produced at steady-state concentrations using Fenton chemistry (H2O2 + Fe2+ ??? Fe3+ + HO- + HO??). Suwannee River fulvic acid and humic acid were used as NOM. The most likely mechanism for the observed inhibition is that hydroxyl radical formation occurs in microenvironmental sites remote from the aromatic compounds. In addition to changes in kinetics, pyrene hydroxyl radical reaction also exhibited a mechanistic change in the presence of fulvic acid. The mechanism changed from a reaction that was apparently firstorder in pyrene to one that was apparently secondorder in pyrene, indicating that pyrene self-reaction may have become the dominant mechanism in the presence of fulvic acid. Dissolved NOM causes significant changes in the rate and mechanism of hydroxyl radical degradation of aromatic compounds. Consequently, literature rate constants measured in pure water will not be useful for predicting the degradation of pollutants in environmental systems. The kinetic and mechanistic information in this study will be useful for developing improved degradation methods involving Fenton chemistry.Reaction of aromatic compounds with hydroxyl radical is inhibited by dissolved natural organic matter (NOM). The degree of inhibition is significantly greater than that expected based on a simple model in which aromatic compounds molecules bounds to NOM are considered to be unreactive. In this study, hydroxyl radical was produced at steady-state concentrations using Fenton chemistry (H2O2 + Fe2+ ??? Fe3+ + HO- + HO??). Suwannee River fulvic acid and humic acid were used as NOM. The most likely mechanisms for the observed inhibition is that hydroxyl radical formation occurs in microenvironmental sites remote from the aromatic compounds. In addition to changes in kinetics, pyrene hydroxyl radical reaction also exhibited a mechanistic change in the presence of fulvic acid. The mechanism changed from a reaction that was apparently first-order in pyrene to one that was apparently second-order in pyrene, indicating that pyrene self-reaction may have become the dominant mechanism in the presence of fulvic acid. Dissolved NOM causes significant changes in the rate and mechanism of hydroxyl radical degradation of aromatic compounds. Consequently, literature rate constants measured in pure water will not be useful for predicting the degradation of pollutants in environmental systems. The kinetic and mechanistic information in this study will be useful for developing improved degradation methods involving Fenton chemistry.

Organic substances in produced and formation water from unconventional natural gas extraction in coal and shale

USGS Publications Warehouse

Orem, William H.; Tatu, Calin A.; Varonka, Matthew S.; Lerch, Harry E.; Bates, Anne L.; Engle, Mark A.; Crosby, Lynn M.; McIntosh, Jennifer

2014-01-01

Organic substances in produced and formation water from coalbed methane (CBM) and gas shale plays from across the USA were examined in this study. Disposal of produced waters from gas extraction in coal and shale is an important environmental issue because of the large volumes of water involved and the variable quality of this water. Organic substances in produced water may be environmentally relevant as pollutants, but have been little studied. Results from five CBM plays and two gas shale plays (including the Marcellus Shale) show a myriad of organic chemicals present in the produced and formation water. Organic compound classes present in produced and formation water in CBM plays include: polycyclic aromatic hydrocarbons (PAHs), heterocyclic compounds, alkyl phenols, aromatic amines, alkyl aromatics (alkyl benzenes, alkyl biphenyls), long-chain fatty acids, and aliphatic hydrocarbons. Concentrations of individual compounds range from < 1 to 100 μg/L, but total PAHs (the dominant compound class for most CBM samples) range from 50 to 100 μg/L. Total dissolved organic carbon (TOC) in CBM produced water is generally in the 1–4 mg/L range. Excursions from this general pattern in produced waters from individual wells arise from contaminants introduced by production activities (oils, grease, adhesives, etc.). Organic substances in produced and formation water from gas shale unimpacted by production chemicals have a similar range of compound classes as CBM produced water, and TOC levels of about 8 mg/L. However, produced water from the Marcellus Shale using hydraulic fracturing has TOC levels as high as 5500 mg/L and a range of added organic chemicals including, solvents, biocides, scale inhibitors, and other organic chemicals at levels of 1000 s of μg/L for individual compounds. Levels of these hydraulic fracturing chemicals and TOC decrease rapidly over the first 20 days of water recovery and some level of residual organic contaminants remain up to 250 days after hydraulic fracturing. Although the environmental impacts of the organics in produced water are not well defined, results suggest that care should be exercised in the disposal and release of produced waters containing these organic substances into the environment because of the potential toxicity of many of these substances.

Alkylation of organic aromatic compounds

DOEpatents

Smith, L.A. Jr.; Arganbright, R.P.; Hearn, D.

1993-09-07

Aromatic compounds are alkylated in a catalytic distillation, wherein the catalyst structure also serves as a distillation component by contacting the aromatic compound with a C[sub 2] to C[sub 10] olefin in the catalyst bed under 0.25 to 50 atmospheres of pressure and at temperatures in the range of 80 C to 500 C, using as the catalyst a molecular sieve characterized as acidic or an acidic cation exchange resin. For example, ethyl benzene is produced by feeding ethylene to about the mid point of the catalyst bed while benzene is conveniently added through the reflux in molar excess to that required to react with ethylene, thereby reacting substantially all of the ethylene and recovering benzene as the principal overhead and ethyl benzene in the bottoms. 1 figures.

Alkylation of organic aromatic compounds

DOEpatents

Smith, Jr., Lawrence A.; Arganbright, Robert P.; Hearn, Dennis

1993-01-01

Aromatic compounds are alkylated in a catalytic distillation, wherein the catalyst structure also serves as a distillation component by contacting the aromatic compound with a C.sub.2 to C.sub.10 olefin in the catalyst bed under 0.25 to 50 atmospheres of pressure and at temperatures in the range of 80.degree. C. to 500.degree. C., using as the catalyst a mole sieve characterized as acidic or an acidic cation exchange resin. For example, ethyl benzene is produced by feeding ethylene to about the mid point of the catalyst bed while benzene is conveniently added through the reflux in molar excess to that required to react with ethylene, thereby reacting substantially all of the ethylene and recovering benzene as the principal overhead and ethyl benzene in the bottoms.

Fuel quality combustion analysis

NASA Technical Reports Server (NTRS)

Naegeli, D. W.; Moses, C. A.

1979-01-01

A high pressure research combustor operating over a wide range of burner inlet conditions was used to determine the effects of fuel molecular structure on soot formation. Six test fuels with equal hydrogen content (12.8%) were blended to stress different molecular components and final boiling points. The fuels containing high concentrations (20%) of polycyclic aromatics and partially saturated polycyclic structures such as tetralin, produced more soot than would be expected from a hydrogen content correlation for typical petroleum based fuels. Fuels containing naphthenes such as decalin agreed with the hydrogen content correlation. The contribution of polycyclic aromatics to soot formation was equivalent to a reduction in fuel hydrogen content of about one percent. The fuel sensitivity to soot formation due to the polycyclic aromatic contribution decreased as burner inlet pressure and fuel/air ratio increased.

Perfluorocyclobutane containing aromatic ether polymers as planarization materials for alternative magnetic media substrates

NASA Astrophysics Data System (ADS)

Perettie, Donald J.; Judy, Jack; Chen, Qixu; Keirstead, Rick

1994-11-01

Perfluorocyclobutane aromatic ether polymers (PFCB) are being researched as planarization materials for alternative magnetic media substrates allowing smoother surfaces for lower head flying recording. The results of current work reported herein have shown that PFCB can be used to affect surfaces on canasite with R(sub A)'s less than 2 nm. In addition, magnetic media can be produced of a quality comparative to that obtained on standard NiP-coated Al as well as that produced on regular canasite with equivalent coercivities at about 1500-1600 Oe and squarenesses of 0.8 or better. In addition to the above magnetic properties the recording performance was excellent with signal-to-noise ratios of planarized media 3.5 dB higher than that on regular canasite.

Mechanochemical Nitration of Aromatic Compounds

NASA Astrophysics Data System (ADS)

Lagoviyer, Oleg S.; Krishtopa, Larisa; Schoenitz, Mirko; Trivedi, Nirupam J.; Dreizin, Edward L.

2018-04-01

Nitration of organic compounds is necessary to produce many energetic materials, such as TNT and nitrocellulose. The conventional nitration process uses a mixture of concentrated sulfuric and nitric acids as nitrating agents and multiple solvents. The chemicals are corrosive and require special handling and disposal procedures. In this study, aromatic nitration has been achieved using solvent-free mechanochemical processing of environmentally benign precursors. Mononitrotoluene was synthesized by milling toluene with sodium nitrate and molybdenum trioxide as a Lewis acid catalyst. Several parameters affecting the desired product yield were identified and varied. A number of byproducts, i.e., dimers of toluene were also produced, but the selectivity was observed to increase with increasing mononitrotoluene yield. Both absolute mononitrotoluene yields and selectivity of its production increased with the increase in the energy transferred to the material from the milling tools.

Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites.

PubMed

Feng, Zhiyang; Kallifidas, Dimitris; Brady, Sean F

2011-08-02

A single gram of soil is predicted to contain thousands of unique bacterial species. The majority of these species remain recalcitrant to standard culture methods, prohibiting their use as sources of unique bioactive small molecules. The cloning and analysis of DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means of exploring the biosynthetic capacity of natural bacterial populations. Environmental DNA libraries contain large reservoirs of bacterial genetic diversity from which new secondary metabolite gene clusters can be systematically recovered and studied. The identification and heterologous expression of type II polyketide synthase-containing eDNA clones is reported here. Functional analysis of three soil DNA-derived polyketide synthase systems in Streptomyces albus revealed diverse metabolites belonging to well-known, rare, and previously uncharacterized structural families. The first of these systems is predicted to encode the production of the known antibiotic landomycin E. The second was found to encode the production of a metabolite with a previously uncharacterized pentacyclic ring system. The third was found to encode the production of unique KB-3346-5 derivatives, which show activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These results, together with those of other small-molecule-directed metagenomic studies, suggest that culture-independent approaches are capable of accessing biosynthetic diversity that has not yet been extensively explored using culture-based methods. The large-scale functional screening of eDNA clones should be a productive strategy for generating structurally previously uncharacterized chemical entities for use in future drug development efforts.

Chip-based polyketide biosynthesis and functionalization.

PubMed

Ku, Bosung; Cha, Junhoe; Srinivasan, Aravind; Kwon, Seok Joon; Jeong, Jae-Choel; Sherman, David H; Dordick, Jonathan S

2006-01-01

We demonstrate construction and novel compound synthesis from a synthetic metabolic pathway consisting of a type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) from Streptomyces coelicolor and soybean peroxidase (SBP) in a microfluidic platform. THNS immobilized to Ni-NTA agarose beads is prepacked into a microfluidic channel, while SBP is covalently attached to the walls of a second microfluidic channel precoated with a reactive poly(maleic anhydride) derivative. The result is a tandem, two-step biochip that enables the synthesis of novel polyketide derivatives. The first microchannel, consisting of THNS, results in the conversion of malonyl-CoA to flaviolin in yields up to 40% with a residence time of 6 min. This conversion is similar to that obtained in several-milliliter batch reactions after 2 h. Linking this microchannel to the SBP microchannel results in biflaviolin synthesis. During the course of this work, we discovered that the substrate specificity of THNS could be manipulated by simply changing the reaction pH. As a result, the starter acyl-CoA specificity can be broadened to yield a series of truncated pyrone products. When combined with variations in the ratio of acyl-CoA and malonyl-CoA (extender substrate) feed rates, high yields of the pyrone products could be achieved, which is further structurally diversified from self- and cross-coupling in the SBP microchannel. The ability to rapidly evaluate the effects of reaction conditions and synthetic multienzyme pathways on a microfludic platform provides a new paradigm for performing metabolic pathway engineering, namely, the reconstruction of pathways for use in new compound discovery.