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

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.

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

The Structural Enzymology of Iterative Aromatic Polyketide Synthases: A Critical Comparison with Fatty Acid Synthases.

PubMed

Tsai, Shiou-Chuan Sheryl

2018-06-20

Polyketides are a large family of structurally complex natural products including compounds with important bioactivities. Polyketides are biosynthesized by polyketide synthases (PKSs), multienzyme complexes derived evolutionarily from fatty acid synthases (FASs). The focus of this review is to critically compare the properties of FASs with iterative aromatic PKSs, including type II PKSs and fungal type I nonreducing PKSs whose chemical logic is distinct from that of modular PKSs. This review focuses on structural and enzymological studies that reveal both similarities and striking differences between FASs and aromatic PKSs. The potential application of FAS and aromatic PKS structures for bioengineering future drugs and biofuels is highlighted.

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

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.

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

Engineering modular polyketide synthases for production of biofuels and industrial chemicals.

PubMed

Cai, Wenlong; Zhang, Wenjun

2018-04-01

Polyketide synthases (PKSs) are one of the most profound biosynthetic factories for producing polyketides with diverse structures and biological activities. These enzymes have been historically studied and engineered to make un-natural polyketides for drug discovery, and have also recently been explored for synthesizing biofuels and industrial chemicals due to their versatility and customizability. Here, we review recent advances in the mechanistic understanding and engineering of modular PKSs for producing polyketide-derived chemicals, and provide perspectives on this relatively new application of PKSs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacterial Genome Adaptation to Niches: Divergence of the Potential Virulence Genes in Three Burkholderia Species of Different Survival Strategies

DTIC Science & Technology

2005-12-01

polyketide synthase ), some TTSS genes (e.g., BMAA1617 putative hrp protein and BMAA1619 hypo- thetical protein), and cell envelope synthesis genes (e.g...License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the...burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters

Genomic and transcriptomic analysis of the endophytic fungus Pestalotiopsis fici reveals its lifestyle and high potential for synthesis of natural products.

PubMed

Wang, Xiuna; Zhang, Xiaoling; Liu, Ling; Xiang, Meichun; Wang, Wenzhao; Sun, Xiang; Che, Yongsheng; Guo, Liangdong; Liu, Gang; Guo, Liyun; Wang, Chengshu; Yin, Wen-Bing; Stadler, Marc; Zhang, Xinyu; Liu, Xingzhong

2015-01-27

In recent years, the genus Pestalotiopsis is receiving increasing attention, not only because of its economic impact as a plant pathogen but also as a commonly isolated endophyte which is an important source of bioactive natural products. Pestalotiopsis fici Steyaert W106-1/CGMCC3.15140 as an endophyte of tea produces numerous novel secondary metabolites, including chloropupukeananin, a derivative of chlorinated pupukeanane that is first discovered in fungi. Some of them might be important as the drug leads for future pharmaceutics. Here, we report the genome sequence of the endophytic fungus of tea Pestalotiopsis fici W106-1/CGMCC3.15140. The abundant carbohydrate-active enzymes especially significantly expanding pectinases allow the fungus to utilize the limited intercellular nutrients within the host plants, suggesting adaptation of the fungus to endophytic lifestyle. The P. fici genome encodes a rich set of secondary metabolite synthesis genes, including 27 polyketide synthases (PKSs), 12 non-ribosomal peptide synthases (NRPSs), five dimethylallyl tryptophan synthases, four putative PKS-like enzymes, 15 putative NRPS-like enzymes, 15 terpenoid synthases, seven terpenoid cyclases, seven fatty-acid synthases, and five hybrids of PKS-NRPS. The majority of these core enzymes distributed into 74 secondary metabolite clusters. The putative Diels-Alderase genes have undergone expansion. The significant expansion of pectinase encoding genes provides essential insight in the life strategy of endophytes, and richness of gene clusters for secondary metabolites reveals high potential of natural products of endophytic fungi.

Molecular Basis for Mycophenolic Acid Biosynthesis in Penicillium brevicompactum▿†

PubMed Central

Regueira, Torsten Bak; Kildegaard, Kanchana Rueksomtawin; Hansen, Bjarne Gram; Mortensen, Uffe H.; Hertweck, Christian; Nielsen, Jens

2011-01-01

Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering. PMID:21398490

Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection.

PubMed

Shevchuk, Olga; Pägelow, Dennis; Rasch, Janine; Döhrmann, Simon; Günther, Gabriele; Hoppe, Julia; Ünal, Can Murat; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel; Steinert, Michael

2014-11-01

L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease. Copyright © 2014 Elsevier GmbH. All rights reserved.

Crystallization and preliminary crystallographic analysis of an acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

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

Morita, Hiroyuki; Kondo, Shin; Kato, Ryohei

2007-07-01

An acridone-producing novel type III polyketide synthase from H. serrata has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 2.0 Å. Polyketide synthase 1 (PKS1) from Huperzia serrata is a plant-specific type III polyketide synthase that shows an unusually versatile catalytic potential, producing various aromatic tetraketides, including chalcones, benzophenones, phlorogulucinols and acridones. Recombinant H. serrata PKS1 expressed in Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 73.3, b = 85.0, c = 137.7 Å, α =more » β = γ = 90.0°. Diffraction data were collected to 2.0 Å resolution using synchrotron radiation at BL24XU of SPring-8.« less

Genome mining of the sordarin biosynthetic gene cluster from Sordaria araneosa Cain ATCC 36386: characterization of cycloaraneosene synthase and GDP-6-deoxyaltrose transferase.

PubMed

Kudo, Fumitaka; Matsuura, Yasunori; Hayashi, Takaaki; Fukushima, Masayuki; Eguchi, Tadashi

2016-07-01

Sordarin is a glycoside antibiotic with a unique tetracyclic diterpene aglycone structure called sordaricin. To understand its intriguing biosynthetic pathway that may include a Diels-Alder-type [4+2]cycloaddition, genome mining of the gene cluster from the draft genome sequence of the producer strain, Sordaria araneosa Cain ATCC 36386, was carried out. A contiguous 67 kb gene cluster consisting of 20 open reading frames encoding a putative diterpene cyclase, a glycosyltransferase, a type I polyketide synthase, and six cytochrome P450 monooxygenases were identified. In vitro enzymatic analysis of the putative diterpene cyclase SdnA showed that it catalyzes the transformation of geranylgeranyl diphosphate to cycloaraneosene, a known biosynthetic intermediate of sordarin. Furthermore, a putative glycosyltransferase SdnJ was found to catalyze the glycosylation of sordaricin in the presence of GDP-6-deoxy-d-altrose to give 4'-O-demethylsordarin. These results suggest that the identified sdn gene cluster is responsible for the biosynthesis of sordarin. Based on the isolated potential biosynthetic intermediates and bioinformatics analysis, a plausible biosynthetic pathway for sordarin is proposed.

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.

An Sfp-type PPTase and associated polyketide and nonribosomal peptide synthases in Agrobacterium vitis are essential for induction of tobacco hypersensitive response and grape necrosis.

PubMed

Zheng, Desen; Burr, Thomas J

2013-07-01

An Sfp-type phosphopantetheinyl transferase (PPTase) encoding gene F-avi5813 in Agrobacterium vitis F2/5 was found to be required for the induction of a tobacco hypersensitive response (HR) and grape necrosis. Sfp-type PPTases are post-translation modification enzymes that activate acyl-carry protein (ACP) domains in polyketide synthases (PKS) and peptidyl-carrier protein (PCP) domains of nonribosomal peptide synthases (NRPS). Mutagenesis of PKS and NRPS genes in A. vitis led to the identification of a PKS gene (F-avi4330) and NRPS gene (F-avi3342) that are both required for HR and necrosis. The gene immediately downstream of F-avi4330 (F-avi4329) encoding a predicted aminotransferase was also found to be required for HR and necrosis. Regulation of F-avi4330 and F-avi3342 by quorum-sensing genes avhR, aviR, and avsR and by a lysR-type regulator, lhnR, was investigated. It was determined that F-avi4330 expression is positively regulated by avhR, aviR, and lhnR and negatively regulated by avsR. F-avi3342 was found to be positively regulated by avhR, aviR, and avsR and negatively regulated by lhnR. Our results suggest that a putative hybrid peptide-polyketide metabolite synthesized by F-avi4330 and F-avi3342 is associated with induction of tobacco HR and grape necrosis. This is the first report that demonstrates that NRPS and PKS play essential roles in conferring the unique ability of A. vitis to elicit a non-host-specific HR and host-specific necrosis.

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...

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.

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.

Aromatic polyketide synthases from 127 Fusarium: pas de deux for chemical diversity

USDA-ARS?s Scientific Manuscript database

Fusarium species collectively cause disease on almost all crop plants and produce numerous natural products (NPs), including mycotoxins, of great concern. Many Fusarium NPs are derived from polyketide synthases (PKSs), large enzymes that catalyze the condensation of simple carboxylic acids. To gain ...

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.

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

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 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.

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.

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.

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.

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

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 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

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

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

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

An Amidinohydrolase Provides the Missing Link in the Biosynthesis of Amino Marginolactone Antibiotics.

PubMed

Hong, Hui; Samborskyy, Markiyan; Lindner, Frederick; Leadlay, Peter F

2016-01-18

Desertomycin A is an aminopolyol polyketide containing a macrolactone ring. We have proposed that desertomycin A and similar compounds (marginolactones) are formed by polyketide synthases primed not with γ-aminobutanoyl-CoA but with 4-guanidinylbutanoyl-CoA, to avoid facile cyclization of the starter unit. This hypothesis requires that there be a final-stage de-amidination of the corresponding guanidino-substituted natural product, but no enzyme for such a process has been described. We have now identified candidate amidinohydrolase genes within the desertomycin and primycin clusters. Deletion of the putative desertomycin amidinohydrolase gene dstH in Streptomyces macronensis led to the accumulation of desertomycin B, the guanidino form of the antibiotic. Also, purified DstH efficiently catalyzed the in vitro conversion of desertomycin B into the A form. Hence this amidinohydrolase furnishes the missing link in this proposed naturally evolved example of protective-group chemistry. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

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.

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.

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

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.

Inhibition Kinetics and Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase†,‡

PubMed Central

Korman, Tyler Paz; Tan, Yuhong; Wong, Justin; Luo, Rui; Tsai, Shiou-Chuan

2008-01-01

Type II polyketides are a class of natural products that include pharmaceutically important aromatic compounds such as the antibiotic tetracycline and antitumor compound doxorubicin. The type II polyketide synthase (PKS) is a complex consisting of 5–10 standalone domains homologous to fatty acid synthase (FAS). Polyketide ketoreductase (KR) provides regio- and stereochemical diversity during the reduction. How the type II polyketide KR specifically reduces only the C9 carbonyl group is not well understood. The cocrystal structures of actinorhodin polyketide ketoreductase (actKR) bound with NADPH or NADP+ and the inhibitor emodin were solved with the wild type and P94L mutant of actKR, revealing the first observation of a bent p-quinone in an enzyme active site. Molecular dynamics simulation help explain the origin of the bent geometry. Extensive screening for in vitro substrates shows that unlike FAS KR, the actKR prefers bicyclic substrates. Inhibition kinetics indicate that actKR follows an ordered Bi Bi mechanism. Together with docking simulations that identified a potential phosphopantetheine binding groove, the structural and functional studies reveal that the C9 specificity is a result of active site geometry and substrate ring constraints. The results lay the foundation for the design of novel aromatic polyketide natural products with different reduction patterns. PMID:18205400

Inhibition Kinetics And Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase

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

Korman, T.P.; Tan, Y.-H.; Wong, J.

Type II polyketides are a class of natural products that include pharmaceutically important aromatic compounds such as the antibiotic tetracycline and antitumor compound doxorubicin. The type II polyketide synthase (PKS) is a complex consisting of 5-10 standalone domains homologous to fatty acid synthase (FAS). Polyketide ketoreductase (KR) provides regio- and stereochemical diversity during the reduction. How the type II polyketide KR specifically reduces only the C9 carbonyl group is not well understood. The cocrystal structures of actinorhodin polyketide ketoreductase (actKR) bound with NADPH or NADP{sup +} and the inhibitor emodin were solved with the wild type and P94L mutant ofmore » actKR, revealing the first observation of a bent p-quinone in an enzyme active site. Molecular dynamics simulation help explain the origin of the bent geometry. Extensive screening for in vitro substrates shows that unlike FAS KR, the actKR prefers bicyclic substrates. Inhibition kinetics indicate that actKR follows an ordered Bi Bi mechanism. Together with docking simulations that identified a potential phosphopantetheine binding groove, the structural and functional studies reveal that the C9 specificity is a result of active site geometry and substrate ring constraints. The results lay the foundation for the design of novel aromatic polyketide natural products with different reduction patterns.« less

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

PubMed Central

Zhang, Wenjun; Ames, Brian D.; Tsai, Shiou-Chuan; Tang, Yi

2006-01-01

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity. PMID:16597959

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.

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.

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.

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

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.

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.

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

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.

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…

Characterization of a gene cluster responsible for the biosynthesis of anticancer agent FK228 in Chromobacterium violaceum No. 968.

PubMed

Cheng, Yi-Qiang; Yang, Min; Matter, Andrea M

2007-06-01

A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates.

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.

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.

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

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

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

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.

Fine Tuning of Antibiotic Activity by a Tailoring Hydroxylase in a Trans-AT Polyketide Synthase Pathway.

PubMed

Mohammad, Hadi H; Connolly, Jack A; Song, Zhongshu; Hothersall, Joanne; Race, Paul R; Willis, Christine L; Simpson, Thomas J; Winn, Peter J; Thomas, Christopher M

2018-04-16

The addition or removal of hydroxy groups modulates the activity of many pharmacologically active biomolecules. It can be integral to the basic biosynthetic factory or result from associated tailoring steps. For the anti-MRSA antibiotic mupirocin, removal of a C8-hydroxy group late in the biosynthetic pathway gives the active pseudomonic acid A. An extra hydroxylation, at C4, occurs in the related but more potent antibiotic thiomarinol A. We report here in vivo and in vitro studies that show that the putative non-haem-iron(II)/α-ketoglutaratedependent dioxygenase TmuB, from the thiomarinol cluster, 4-hydroxylates various pseudomonic acids whereas C8-OH, and other substituents around the tetrahydropyran ring, block enzyme action but not substrate binding. Molecular modelling suggested a basis for selectivity, but mutation studies had a limited ability to rationally modify TmuB substrate specificity. 4-Hydroxylation had opposite effects on the potency of mupirocin and thiomarinol. Thus, TmuB can be added to the toolbox of polyketide tailoring technologies for the in vivo generation of new antibiotics in the future. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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 ...

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

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.

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

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.

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.

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.

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

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

Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming

PubMed Central

2015-01-01

Iterative, nonreducing polyketide synthases (NR-PKSs) are multidomain enzymes responsible for the construction of the core architecture of aromatic polyketide natural products in fungi. Engineering these enzymes for the production of non-native metabolites has been a long-standing goal. We conducted a systematic survey of in vitro “domain swapped” NR-PKSs using an enzyme deconstruction approach. The NR-PKSs were dissected into mono- to multidomain fragments and recombined as noncognate pairs in vitro, reconstituting enzymatic activity. The enzymes used in this study produce aromatic polyketides that are representative of the four main chemical features set by the individual NR-PKS: starter unit selection, chain-length control, cyclization register control, and product release mechanism. We found that boundary conditions limit successful chemistry, which are dependent on a set of underlying enzymatic mechanisms. Crucial for successful redirection of catalysis, the rate of productive chemistry must outpace the rate of spontaneous derailment and thioesterase-mediated editing. Additionally, all of the domains in a noncognate system must interact efficiently if chemical redirection is to proceed. These observations refine and further substantiate current understanding of the mechanisms governing NR-PKS catalysis. PMID:24815013

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

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.

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...

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

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.

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.

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

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.

Plant pathogenic anaerobic bacteria use aromatic polyketides to access aerobic territory.

PubMed

Shabuer, Gulimila; Ishida, Keishi; Pidot, Sacha J; Roth, Martin; Dahse, Hans-Martin; Hertweck, Christian

2015-11-06

Around 25% of vegetable food is lost worldwide because of infectious plant diseases, including microbe-induced decay of harvested crops. In wet seasons and under humid storage conditions, potato tubers are readily infected and decomposed by anaerobic bacteria (Clostridium puniceum). We found that these anaerobic plant pathogens harbor a gene locus (type II polyketide synthase) to produce unusual polyketide metabolites (clostrubins) with dual functions. The clostrubins, which act as antibiotics against other microbial plant pathogens, enable the anaerobic bacteria to survive an oxygen-rich plant environment. Copyright © 2015, American Association for the Advancement of Science.

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.

Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining.

PubMed

Tang, Xiaoyu; Li, Jie; Millán-Aguiñaga, Natalie; Zhang, Jia Jia; O'Neill, Ellis C; Ugalde, Juan A; Jensen, Paul R; Mantovani, Simone M; Moore, Bradley S

2015-12-18

Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or "orphaned" secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs and (ii) how to rapidly connect genes to biosynthesized small molecules. Here, we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes colocalized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related ttm BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and autoresistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized but also paves the way for the investigation of novel enzymology involved in thiotetronic acid natural product biosynthesis.

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

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.

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

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.

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

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.

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.

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.

Light Remodels Lipid Biosynthesis in Nannochloropsis gaditana by Modulating Carbon Partitioning between Organelles1[OPEN

PubMed Central

Vitulo, Nicola; Diretto, Gianfranco; Block, Maryse; Jouhet, Juliette; Meneghesso, Andrea; Valle, Giorgio; Giuliano, Giovanni; Maréchal, Eric

2016-01-01

The seawater microalga Nannochloropsis gaditana is capable of accumulating a large fraction of reduced carbon as lipids. To clarify the molecular bases of this metabolic feature, we investigated light-driven lipid biosynthesis in Nannochloropsis gaditana cultures combining the analysis of photosynthetic functionality with transcriptomic, lipidomic and metabolomic approaches. Light-dependent alterations are observed in amino acid, isoprenoid, nucleic acid, and vitamin biosynthesis, suggesting a deep remodeling in the microalgal metabolism triggered by photoadaptation. In particular, high light intensity is shown to affect lipid biosynthesis, inducing the accumulation of diacylglyceryl-N,N,N-trimethylhomo-Ser and triacylglycerols, together with the up-regulation of genes involved in their biosynthesis. Chloroplast polar lipids are instead decreased. This situation correlates with the induction of genes coding for a putative cytosolic fatty acid synthase of type 1 (FAS1) and polyketide synthase (PKS) and the down-regulation of the chloroplast fatty acid synthase of type 2 (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments, favoring the cytoplasm, mitochondrion, and endoplasmic reticulum at the expense of the chloroplast. These results highlight the high flexibility of lipid biosynthesis in N. gaditana and lay the foundations for a hypothetical mechanism of regulation of primary carbon partitioning by controlling metabolite allocation at the subcellular level. PMID:27325666

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.

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

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

Regulation of melanin biosynthesis via the dihydroxynaphthalene pathway is dependent on sexual development in the ascomycete Sordaria macrospora.

PubMed

Engh, Ines; Nowrousian, Minou; Kück, Ulrich

2007-10-01

The filamentous ascomycete Sordaria macrospora accumulates melanin during sexual development. The four melanin biosynthesis genes pks, teh, sdh and tih were isolated and their homology to genes involved in 1,8 dihydroxynaphthalene (DHN) melanin biosynthesis was shown. The presence of DHN melanin in S. macrospora was further confirmed by disrupting the pks gene encoding a putative polyketide synthase and by RNA interference-mediated silencing of the sdh gene encoding a putative scytalone dehydratase. Because melanin occurs in fruiting bodies that develop through several intermediate stages within 7 days of growth, a Northern analysis of a developmental time-course was conducted. These data revealed a time-dependent regulation of teh and sdh transcript levels. Comparing the transcriptional expression by real-time PCR of melanin biosynthesis genes in the wild type under conditions allowing or repressing sexual development, a significant downregulation during vegetative growth was detected. Quantitative real-time PCR and Northern blot analysis of melanin biosynthesis gene expression in different developmental mutants confirmed that melanin biosynthesis is linked to fruiting body development and is under the control of specific regulatory genes that participate in sexual differentiation.

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.

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

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.

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

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.

Genomic Patterns of Pathogen Evolution Revealed by Comparison of Burkholderia pseudomallei, the Causative Agent of Melioidosis, to Avirulent Burkholderia thailandensis

DTIC Science & Technology

2006-05-26

are four polyketide synthase (PKS) and nonribos- omal pepeide synthase (NRPS) clusters involved in the production and regulation of secondary...specific genomic regions, we derived molecular explanations for previously-known metabolic differences, discovered potentially new ones , and found that...Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium

Continuing Evolution of Burkholderia mallei Through Genome Reduction and Large-Scale Rearrangements

DTIC Science & Technology

2010-01-22

in Materials and Methods. b NRPS, nonribosomal peptide synthase ; PKS, polyketide synthase ; RND, resistance nodulation-division like pump. Losada et al...genomics, genome erosion, bacterial virulence. ª The Author(s) 2010. Published by Oxford University Press on behalf of the Society for Molecular Biology...creativecommons.org/licenses/by-nc/ 2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original

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.

Antimicrobial assay and genetic screening of selected freshwater Cyanobacteria and identification of a biomolecule dihydro-2H-pyran-2-one derivative.

PubMed

Srivastava, A; Singh, V K; Patnaik, S; Tripathi, J; Singh, P; Nath, G; Asthana, R K

2017-04-01

Explorations of freshwater Cyanobacteria as antimicrobial (bacteria, fungi and methicillin-resistant Staphylococcus aureus (MRSA) strains) drug resource using bioassay, NRPS (non-ribosomal polypeptide synthetase) and PKS (polyketide synthase) genes, as well as in silico approach. We have bioassayed the extracts of Phormidium CCC727, Geitlerinema CCC728, Arthrospira CCC729, Leptolyngbya CCC732, Phormidium CCC730, Phormidium CCC731 against six pathogenic bacteria comprising Gram (+ve): S. aureus including seven clinical MRSA and Enterococcus faecalis, Gram (-ve): Escherichia coli, Salmonella Typhimurium, Klebsiella pneumoniae and Shigella boydii along with non-pathogenic Enterobacter aerogenes as well as fungal strains (Cryptococcus neoformans and Candida albicans, C. krusei, C. tropicalis and Aspergillus niger) exhibiting antimicrobial potential. The NRPS and PKS genes of the target strains were also amplified and sequenced. The putative protein structures were predicted using bioinformatics approach. PKS gene expression indicated β keto-acyl synthase as one of the important active domains in the biomolecules related to antitumour and antifungal group. The simultaneous identification of the biomolecule (dihydro-2H-pyran-2-one derivative) was also inferred spectroscopically. Freshwater Cyanobacteria are prolific producers of secondary metabolite(s) that may act as the antimicrobial drug resource in addition to their much explored marine counterpart. © 2016 The Society for Applied Microbiology.

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.

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.

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.

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

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

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

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.

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

Huang, Tingting; Chang, Chin -Yuan; Lohman, Jeremy R.

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us tomore » propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. In conclusion, these findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis.« less

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

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

Genetic basis for mycophenolic acid production and strain-dependent production variability in Penicillium roqueforti.

PubMed

Gillot, Guillaume; Jany, Jean-Luc; Dominguez-Santos, Rebeca; Poirier, Elisabeth; Debaets, Stella; Hidalgo, Pedro I; Ullán, Ricardo V; Coton, Emmanuel; Coton, Monika

2017-04-01

Mycophenolic acid (MPA) is a secondary metabolite produced by various Penicillium species including Penicillium roqueforti. The MPA biosynthetic pathway was recently described in Penicillium brevicompactum. In this study, an in silico analysis of the P. roqueforti FM164 genome sequence localized a 23.5-kb putative MPA gene cluster. The cluster contains seven genes putatively coding seven proteins (MpaA, MpaB, MpaC, MpaDE, MpaF, MpaG, MpaH) and is highly similar (i.e. gene synteny, sequence homology) to the P. brevicompactum cluster. To confirm the involvement of this gene cluster in MPA biosynthesis, gene silencing using RNA interference targeting mpaC, encoding a putative polyketide synthase, was performed in a high MPA-producing P. roqueforti strain (F43-1). In the obtained transformants, decreased MPA production (measured by LC-Q-TOF/MS) was correlated to reduced mpaC gene expression by Q-RT-PCR. In parallel, mycotoxin quantification on multiple P. roqueforti strains suggested strain-dependent MPA-production. Thus, the entire MPA cluster was sequenced for P. roqueforti strains with contrasted MPA production and a 174bp deletion in mpaC was observed in low MPA-producers. PCRs directed towards the deleted region among 55 strains showed an excellent correlation with MPA quantification. Our results indicated the clear involvement of mpaC gene as well as surrounding cluster in P. roqueforti MPA biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recognition of extended linear and cyclised polyketide mimics by a type II acyl carrier protein† †Electronic supplementary information (ESI) available: Detailed experimental procedures and characterisation data for all new compounds, additional spectra and structural statistics for derivatised ACP three-dimensional structures. See DOI: 10.1039/c5sc03864b Click here for additional data file.

PubMed Central

Dong, Xu; Bailey, Christopher D.; Williams, Christopher; Crosby, John; Simpson, Thomas J.

2016-01-01

Polyketides are secondary metabolites which display both valuable pharmaceutical and agrochemical properties. Biosynthesis is performed by polyketide synthases (PKSs), and the acyl carrier protein (ACP), a small acidic protein, that transports the growing polyketide chain and is essential for activity. Here we report the synthesis of two aromatic probes and a linear octaketide mimic that have been tethered to actinorhodin ACP. These experiments were aimed at probing the ACP's capacity to sequester a non-polar versus a phenolic aromatic ring (that more closely mimics a polyketide intermediate) as well as investigations with extended polyketide chain surrogates. The binding of these mimics has been assessed using high-resolution solution NMR studies and high-resolution structure determination. These results reveal that surprisingly a PKS ACP is able to bind and sequester a bulky non-polar substrate containing an aromatic ring in a fatty acid type binding mode, but the introduction of even a small degree of polarity favours a markedly different association at a surface site that is distinct from that employed by fatty acid ACPs. PMID:28936328

Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases1

PubMed Central

Pandith, Shahzad A.; Dhar, Niha; Bhat, Wajid Waheed; Kushwaha, Manoj; Gupta, Ajai P.; Shah, Manzoor A.; Vishwakarma, Ram

2016-01-01

Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and their relative substrate selectivities. PMID:27268960

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877

PubMed Central

Bunet, Robert; Riclea, Ramona; Laureti, Luisa; Hôtel, Laurence; Paris, Cédric; Girardet, Jean-Michel; Spiteller, Dieter; Dickschat, Jeroen S.; Leblond, Pierre; Aigle, Bertrand

2014-01-01

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones. PMID:24498152

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...

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.

Enhancement of anti-inflammatory activity of Aloe vera adventitious root extracts through the alteration of primary and secondary metabolites via salicylic acid elicitation.

PubMed

Lee, Yun Sun; Ju, Hyun Kyoung; Kim, Yeon Jeong; Lim, Tae-Gyu; Uddin, Md Romij; Kim, Yeon Bok; Baek, Jin Hong; Kwon, Sung Won; Lee, Ki Won; Seo, Hak Soo; Park, Sang Un; Yang, Tae-Jin

2013-01-01

Aloe vera (Asphodeloideae) is a medicinal plant in which useful secondary metabolites are plentiful. Among the representative secondary metabolites of Aloe vera are the anthraquinones including aloe emodin and chrysophanol, which are tricyclic aromatic quinones synthesized via a plant-specific type III polyketide biosynthesis pathway. However, it is not yet clear which cellular responses can induce the pathway, leading to production of tricyclic aromatic quinones. In this study, we examined the effect of endogenous elicitors on the type III polyketide biosynthesis pathway and identified the metabolic changes induced in elicitor-treated Aloe vera adventitious roots. Salicylic acid, methyl jasmonate, and ethephon were used to treat Aloe vera adventitious roots cultured on MS liquid media with 0.3 mg/L IBA for 35 days. Aloe emodin and chrysophanol were remarkably increased by the SA treatment, more than 10-11 and 5-13 fold as compared with untreated control, respectively. Ultra-performance liquid chromatography-electrospray ionization mass spectrometry analysis identified a total of 37 SA-induced compounds, including aloe emodin and chrysophanol, and 3 of the compounds were tentatively identified as tricyclic aromatic quinones. Transcript accumulation analysis of polyketide synthase genes and gas chromatography mass spectrometry showed that these secondary metabolic changes resulted from increased expression of octaketide synthase genes and decreases in malonyl-CoA, which is the precursor for the tricyclic aromatic quinone biosynthesis pathway. In addition, anti-inflammatory activity was enhanced in extracts of SA-treated adventitious roots. Our results suggest that SA has an important role in activation of the plant specific-type III polyketide biosynthetic pathway, and therefore that the efficacy of Aloe vera as medicinal agent can be improved through SA treatment.

Enhancement of Anti-Inflammatory Activity of Aloe vera Adventitious Root Extracts through the Alteration of Primary and Secondary Metabolites via Salicylic Acid Elicitation

PubMed Central

Lee, Yun Sun; Ju, Hyun Kyoung; Kim, Yeon Jeong; Lim, Tae-Gyu; Uddin, Md Romij; Kim, Yeon Bok; Baek, Jin Hong; Kwon, Sung Won; Lee, Ki Won; Seo, Hak Soo; Park, Sang Un; Yang, Tae-Jin

2013-01-01

Aloe vera (Asphodeloideae) is a medicinal plant in which useful secondary metabolites are plentiful. Among the representative secondary metabolites of Aloe vera are the anthraquinones including aloe emodin and chrysophanol, which are tricyclic aromatic quinones synthesized via a plant-specific type III polyketide biosynthesis pathway. However, it is not yet clear which cellular responses can induce the pathway, leading to production of tricyclic aromatic quinones. In this study, we examined the effect of endogenous elicitors on the type III polyketide biosynthesis pathway and identified the metabolic changes induced in elicitor-treated Aloe vera adventitious roots. Salicylic acid, methyl jasmonate, and ethephon were used to treat Aloe vera adventitious roots cultured on MS liquid media with 0.3 mg/L IBA for 35 days. Aloe emodin and chrysophanol were remarkably increased by the SA treatment, more than 10–11 and 5–13 fold as compared with untreated control, respectively. Ultra-performance liquid chromatography-electrospray ionization mass spectrometry analysis identified a total of 37 SA-induced compounds, including aloe emodin and chrysophanol, and 3 of the compounds were tentatively identified as tricyclic aromatic quinones. Transcript accumulation analysis of polyketide synthase genes and gas chromatography mass spectrometry showed that these secondary metabolic changes resulted from increased expression of octaketide synthase genes and decreases in malonyl-CoA, which is the precursor for the tricyclic aromatic quinone biosynthesis pathway. In addition, anti-inflammatory activity was enhanced in extracts of SA-treated adventitious roots. Our results suggest that SA has an important role in activation of the plant specific-type III polyketide biosynthetic pathway, and therefore that the efficacy of Aloe vera as medicinal agent can be improved through SA treatment. PMID:24358188

Aromatic Polyketide GTRI-02 is a Previously Unidentified Product of the act Gene Cluster in Streptomyces coelicolor A3(2).

PubMed

Wu, Changsheng; Ichinose, Koji; Choi, Young Hae; van Wezel, Gilles P

2017-07-18

The biosynthesis of aromatic polyketides derived from type II polyketide synthases (PKSs) is complex, and it is not uncommon that highly similar gene clusters give rise to diverse structural architectures. The act biosynthetic gene cluster (BGC) of the model actinomycete Streptomyces coelicolor A3(2) is an archetypal type II PKS. Here we show that the act BGC also specifies the aromatic polyketide GTRI-02 (1) and propose a mechanism for the biogenesis of its 3,4-dihydronaphthalen-1(2H)-one backbone. Polyketide 1 was also produced by Streptomyces sp. MBT76 after activation of the act-like qin gene cluster by overexpression of the pathway-specific activator. Mining of this strain also identified dehydroxy-GTRI-02 (2), which most likely originated from dehydration of 1 during the isolation process. This work shows that even extensively studied model gene clusters such as act of S. coelicolor can still produce new chemistry, offering new perspectives for drug discovery. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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).

The Janthinobacterium sp. HH01 Genome Encodes a Homologue of the V. cholerae CqsA and L. pneumophila LqsA Autoinducer Synthases

PubMed Central

Hornung, Claudia; Poehlein, Anja; Haack, Frederike S.; Schmidt, Martina; Dierking, Katja; Pohlen, Andrea; Schulenburg, Hinrich; Blokesch, Melanie; Plener, Laure; Jung, Kirsten; Bonge, Andreas; Krohn-Molt, Ines; Utpatel, Christian; Timmermann, Gabriele; Spieck, Eva; Pommerening-Röser, Andreas; Bode, Edna; Bode, Helge B.; Daniel, Rolf; Schmeisser, Christel; Streit, Wolfgang R.

2013-01-01

Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes. PMID:23405110

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 ...

Structure and Absolute Configuration of Jurassic Polyketide-Derived Spiroborate Pigments Obtained from Microgram Quantities.

PubMed

Wolkenstein, Klaus; Sun, Han; Falk, Heinz; Griesinger, Christian

2015-10-28

Complete structural elucidation of natural products is often challenging due to structural complexity and limited availability. This is true for present-day secondary metabolites, but even more for exceptionally preserved secondary metabolites of ancient organisms that potentially provide insights into the evolutionary history of natural products. Here, we report the full structure and absolute configuration of the borolithochromes, enigmatic boron-containing pigments from a Jurassic putative red alga, from samples of less than 50 μg using microcryoprobe NMR, circular dichroism spectroscopy, and density functional theory calculations and reveal their polyketide origin. The pigments are identified as spiroborates with two pentacyclic sec-butyl-trihydroxy-methyl-benzo[gh]tetraphen-one ligands and less-substituted derivatives. The configuration of the sec-butyl group is found to be (S). Because the exceptional benzo[gh]tetraphene scaffold is otherwise only observed in the recently discovered polyketide clostrubin from a present-day Clostridium bacterium, the Jurassic borolithochromes now can be unambiguously linked to the modern polyketide, providing evidence that the fossil pigments are almost originally preserved secondary metabolites and suggesting that the pigments in fact may have been produced by an ancient bacterium. The borolithochromes differ fundamentally from previously described boronated polyketides and represent the first boronated aromatic polyketides found so far. Our results demonstrate the potential of microcryoprobe NMR in the analysis of previously little-explored secondary metabolites from ancient organisms and reveal the evolutionary significance of clostrubin-type polyketides.

Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Biowarfare Simulant

DTIC Science & Technology

2011-03-25

379 1317617 BG1320 (06415) NS pksR – Polyketide synthase BSU17720 (71.0) G:C C:S 1698/2574 1326096 BG1327 (06450) NS ebrB – multidrug resistance...frameshift mutation in the mmgD gene on the C-terminus of the 2-methylcitrate synthase homolog of B. atrophaeus strain Detrick-1. Arrow indicates the...lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole

Uncovering the formation and selection of benzylmalonyl-CoA from the biosynthesis of splenocin and enterocin reveals a versatile way to introduce amino acids into polyketide carbon scaffolds.

PubMed

Chang, Chenchen; Huang, Rong; Yan, Yan; Ma, Hongmin; Dai, Zheng; Zhang, Benying; Deng, Zixin; Liu, Wen; Qu, Xudong

2015-04-01

Selective modification of carbon scaffolds via biosynthetic engineering is important for polyketide structural diversification. Yet, this scope is currently restricted to simple aliphatic groups due to (1) limited variety of CoA-linked extender units, which lack aromatic structures and chemical reactivity, and (2) narrow acyltransferase (AT) specificity, which is limited to aliphatic CoA-linked extender units. In this report, we uncovered and characterized the first aromatic CoA-linked extender unit benzylmalonyl-CoA from the biosynthetic pathways of splenocin and enterocin in Streptomyces sp. CNQ431. Its synthesis employs a deamination/reductive carboxylation strategy to convert phenylalanine into benzylmalonyl-CoA, providing a link between amino acid and CoA-linked extender unit synthesis. By characterization of its selection, we further validated that AT domains of splenocin, and antimycin polyketide synthases are able to select this extender unit to introduce the phenyl group into their dilactone scaffolds. The biosynthetic machinery involved in the formation of this extender unit is highly versatile and can be potentially tailored for tyrosine, histidine and aspartic acid. The disclosed aromatic extender unit, amino acid-oriented synthetic pathway, and aromatic-selective AT domains provides a systematic breakthrough toward current knowledge of polyketide extender unit formation and selection, and also opens a route for further engineering of polyketide carbon scaffolds using amino acids.

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.

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

DOE PAGES

Huang, Tingting; Chang, Chin -Yuan; Lohman, Jeremy R.; ...

2016-10-01

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us tomore » propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. In conclusion, these findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis.« less

Investigation of biotechnological potential of sponge-associated bacteria collected in Brazilian coast.

PubMed

Santos, O C S; Soares, A R; Machado, F L S; Romanos, M T V; Muricy, G; Giambiagi-deMarval, M; Laport, M S

2015-02-01

Marine bacteria are a rich source of structurally unique natural compounds, several of which have shown a wide variety of biological activities. In this study, the metabolites present in the culture supernatants of the eight sponge-associated bacteria were extracted using ethyl acetate, and all extracts showed activity against Staphylococcus aureus. Subsequently, the extracts of the Pseudomonas fluorescens H40 and H41, and Pseudomonas aeruginosa H51 were subjected to solvent partitioning, and the active fractions were submitted to chromatographic separation. Three different active fractions were obtained, one of which was identified as diketopiperazine cyclo-(L-Leu-L-Pro). This substance was bactericidal for Staph. aureus and Ps. aeruginosa and showed cytotoxic activity against HEp-2 tumour cells. Putative gene fragments coding for the type I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) domains were PCR-amplified from five and three strains, respectively. The results suggest that sponge-associated bacteria analysed in this study may represent a potential source for production of antimicrobial substances against bacterial pathogens of medical importance. © 2014 The Society for Applied Microbiology.

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

SeMPI: a genome-based secondary metabolite prediction and identification web server.

PubMed

Zierep, Paul F; Padilla, Natàlia; Yonchev, Dimitar G; Telukunta, Kiran K; Klementz, Dennis; Günther, Stefan

2017-07-03

The secondary metabolism of bacteria, fungi and plants yields a vast number of bioactive substances. The constantly increasing amount of published genomic data provides the opportunity for an efficient identification of gene clusters by genome mining. Conversely, for many natural products with resolved structures, the encoding gene clusters have not been identified yet. Even though genome mining tools have become significantly more efficient in the identification of biosynthetic gene clusters, structural elucidation of the actual secondary metabolite is still challenging, especially due to as yet unpredictable post-modifications. Here, we introduce SeMPI, a web server providing a prediction and identification pipeline for natural products synthesized by polyketide synthases of type I modular. In order to limit the possible structures of PKS products and to include putative tailoring reactions, a structural comparison with annotated natural products was introduced. Furthermore, a benchmark was designed based on 40 gene clusters with annotated PKS products. The web server of the pipeline (SeMPI) is freely available at: http://www.pharmaceutical-bioinformatics.de/sempi. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

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

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

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis.

PubMed

Xie, Xinqiang; Garg, Ashish; Khosla, Chaitan; Cane, David E

2017-03-01

The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR 0 ) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR 0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2- 2 H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP + in the presence of redox-inactive, recombinant NanKR1 0 or NanKR5 0 , from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR7 0 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR 0 -catalyzed isotope exchange of the reversibly generated, transiently formed oxidation product [2- 2 H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR 0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR 0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.

Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis

PubMed Central

Xie, Xinqiang; Garg, Ashish; Khosla, Chaitan; Cane, David E.

2017-01-01

The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin (1) and salinomycin (4) harbor a number of redox-inactive ketoreductase (KR0) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0 domains is, as predicted, a (2R)-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S-propionyl-N-acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-2H]-(2R,3S)-2-methyl-3-hydroxypentanoyl-ACP (6a) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP+ in the presence of redox-inactive, recombinant NanKR10 or NanKR50, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR70 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a. Control experiments confirmed that this washout was due to KR0-catalyzed isotope exchange of the reversibly-generated, transiently-formed oxidation product [2-2H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations. PMID:28157306

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.

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

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.

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

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...

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...

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.

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.

Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple ‘Holsteiner Cox’ 1[W][OA

PubMed Central

Chizzali, Cornelia; Gaid, Mariam M.; Belkheir, Asma K.; Hänsch, Robert; Richter, Klaus; Flachowsky, Henryk; Peil, Andreas; Hanke, Magda-Viola; Liu, Benye; Beerhues, Ludger

2012-01-01

Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple ‘Golden Delicious’, nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple ‘Holsteiner Cox,’ heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple ‘Cox Orange,’ expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells. PMID:22158676

The Cer-cqu gene cluster determines three key players in a β-diketone synthase polyketide pathway synthesizing aliphatics in epicuticular waxes.

PubMed

Schneider, Lizette M; Adamski, Nikolai M; Christensen, Caspar Elo; Stuart, David B; Vautrin, Sonia; Hansson, Mats; Uauy, Cristobal; von Wettstein-Knowles, Penny

2016-03-09

Aliphatic compounds on plant surfaces, called epicuticular waxes, are the first line of defense against pathogens and pests, contribute to reducing water loss and determine other important phenotypes. Aliphatics can form crystals affecting light refraction, resulting in a color change and allowing identification of mutants in their synthesis or transport. The present study discloses three such Eceriferum (cer) genes in barley - Cer-c, Cer-q and Cer-u - known to be tightly linked and functioning in a biochemical pathway forming dominating amounts of β-diketone and hydroxy-β-diketones plus some esterified alkan-2-ols. These aliphatics are present in many Triticeae as well as dicotyledons such as Eucalyptus and Dianthus. Recently developed genomic resources and mapping populations in barley defined these genes to a small region on chromosome arm 2HS. Exploiting Cer-c and -u potential functions pinpointed five candidates, of which three were missing in apparent cer-cqu triple mutants. Sequencing more than 50 independent mutants for each gene confirmed their identification. Cer-c is a chalcone synthase-like polyketide synthase, designated diketone synthase (DKS), Cer-q is a lipase/carboxyl transferase and Cer-u is a P450 enzyme. All were highly expressed in pertinent leaf sheath tissue of wild type. A physical map revealed the order Cer-c, Cer-u, Cer-q with the flanking genes 101kb apart, confirming they are a gene cluster, Cer-cqu. Homology-based modeling suggests that many of the mutant alleles affect overall protein structure or specific active site residues. The rich diversity of identified mutations will facilitate future studies of three key enzymes involved in synthesis of plant apoplast waxes. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The Cer-cqu gene cluster determines three key players in a β-diketone synthase polyketide pathway synthesizing aliphatics in epicuticular waxes

PubMed Central

Schneider, Lizette M; Adamski, Nikolai M; Christensen, Caspar Elo; Stuart, David B; Vautrin, Sonia; Hansson, Mats; Uauy, Cristobal; von Wettstein-Knowles, Penny

2016-01-01

Aliphatic compounds on plant surfaces, called epicuticular waxes, are the first line of defense against pathogens and pests, contribute to reducing water loss and determine other important phenotypes. Aliphatics can form crystals affecting light refraction, resulting in a color change and allowing identification of mutants in their synthesis or transport. The present study discloses three such Eceriferum (cer) genes in barley – Cer-c, Cer-q and Cer-u – known to be tightly linked and functioning in a biochemical pathway forming dominating amounts of β-diketone and hydroxy-β-diketones plus some esterified alkan-2-ols. These aliphatics are present in many Triticeae as well as dicotyledons such as Eucalyptus and Dianthus. Recently developed genomic resources and mapping populations in barley defined these genes to a small region on chromosome arm 2HS. Exploiting Cer-c and -u potential functions pinpointed five candidates, of which three were missing in apparent cer-cqu triple mutants. Sequencing more than 50 independent mutants for each gene confirmed their identification. Cer-c is a chalcone synthase-like polyketide synthase, designated diketone synthase (DKS), Cer-q is a lipase/carboxyl transferase and Cer-u is a P450 enzyme. All were highly expressed in pertinent leaf sheath tissue of wild type. A physical map revealed the order Cer-c, Cer-u, Cer-q with the flanking genes 101kb apart, confirming they are a gene cluster, Cer-cqu. Homology-based modeling suggests that many of the mutant alleles affect overall protein structure or specific active site residues. The rich diversity of identified mutations will facilitate future studies of three key enzymes involved in synthesis of plant apoplast waxes. PMID:26962211

Plant polyketide synthases: a chalcone synthase-type enzyme which performs a condensation reaction with methylmalonyl-CoA in the biosynthesis of C-methylated chalcones.

PubMed

Schröder, J; Raiber, S; Berger, T; Schmidt, A; Schmidt, J; Soares-Sello, A M; Bardshiri, E; Strack, D; Simpson, T J; Veit, M; Schröder, G

1998-06-09

Heterologous screening of a cDNA library from Pinusstrobus seedlings identified clones for two chalcone synthase (CHS) related proteins (PStrCHS1 and PStrCHS2, 87.6% identity). Heterologous expression in Escherichia coli showed that PStrCHS1 performed the typical CHS reaction, that it used starter CoA-esters from the phenylpropanoid pathway, and that it performed three condensation reactions with malonyl-CoA, followed by the ring closure to the chalcone. PstrCHS2 was completely inactive with these starters and also with linear CoA-esters. Activity was detected only with a diketide derivative (N-acetylcysteamine thioester of 3-oxo-5-phenylpent-4-enoic acid) that corresponded to the CHS reaction intermediate postulated after the first condensation reaction. PstrCHS2 performed only one condensation, with 6-styryl-4-hydroxy-2-pyrone derivatives as release products. The enzyme preferred methylmalonyl-CoA against malonyl-CoA, if only methylmalonyl-CoA was available. These properties and a comparison with the CHS from Pinus sylvestris suggested for PstrCHS2 a special function in the biosynthesis of secondary products. In contrast to P. sylvestris, P. strobus contains C-methylated chalcone derivatives, and the methyl group is at the position predicted from a chain extension with methylmalonyl-CoA in the second condensation of the biosynthetic reaction sequence. We propose that PstrCHS2 specifically contributes the condensing reaction with methylmalonyl-CoA to yield a methylated triketide intermediate. We discuss a model that the biosynthesis of C-methylated chalcones represents the simplest example of a modular polyketide synthase.

A polyketide synthase gene and an aspartate kinase like gene are required for the biosynthesis of fusaric acid in Fusarium Oxysporum f. sp. Vasinfectum

USDA-ARS?s Scientific Manuscript database

A genetically unique strain of the Fusarium wilt pathogen was first recognized in wilted and dead Upland cotton seedlings in Australia in 1993. Since that time the disease spread rapidly despite stringent containment practices. The Australian biotype isolates of Fusarium oxysporum f. sp. vasinfec...

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.

Structural and Functional Studies of a Pyran Synthase Domain from a trans-Acyltransferase Assembly Line.

PubMed

Wagner, Drew T; Zhang, Zhicheng; Meoded, Roy A; Cepeda, Alexis J; Piel, Jörn; Keatinge-Clay, Adrian T

2018-04-20

trans-Acyltransferase assembly lines possess enzymatic domains often not observed in their better characterized cis-acyltransferase counterparts. Within this repertoire of largely unexplored biosynthetic machinery is a class of enzymes called the pyran synthases that catalyze the formation of five- and six-membered cyclic ethers from diverse polyketide chains. The 1.55 Å resolution crystal structure of a pyran synthase domain excised from the ninth module of the sorangicin assembly line highlights the similarity of this enzyme to the ubiquitous dehydratase domain and provides insight into the mechanism of ring formation. Functional assays of point mutants reveal the central importance of the active site histidine that is shared with the dehydratases as well as the supporting role of a neighboring semiconserved asparagine.

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.

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.

Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.

PubMed

Siméone, Roxane; Constant, Patricia; Guilhot, Christophe; Daffé, Mamadou; Chalut, Christian

2007-07-01

Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.

The genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors

PubMed Central

Schorn, Michelle; Zettler, Judith; Noel, Joseph P.; Dorrestein, Pieter C.; Moore, Bradley S.; Kaysser, Leonard

2013-01-01

The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α′,β′-epoxyketone pharmacophore allows binding to the catalytic β-subunits of the proteasome with extraordinary specificity. Here we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epoxyketones. The clusters for epoxomicin, the lead compound for the anti-cancer drug Kyprolis™, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid non-ribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation. PMID:24168704

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

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

Preparation of a Burkholderia Mallei Vaccine

DTIC Science & Technology

2000-01-01

together with the indications of the portions of this data which are subject to such limitations, shall be included on any reproduction hereof which... on to apoptosis; hence, virulent mycobacteria will survive in those macrophages. To assess any similarity between Mycobacterium and Burkholderia...the presence of an open reading frame encoding for a type I polyketide synthase from Streptomyces species (data not 13 shown). We are currently

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.

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli.

PubMed

Xie, Xi; Meesapyodsuk, Dauenpen; Qiu, Xiao

2017-05-01

Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. coli fabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Thraustochytrium IMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protist Thraustochytrium sp. strain ATCC 26185 and then expressing them in wild-type E. coli and mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes in E. coli This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs. Copyright © 2017 American Society for Microbiology.

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli

PubMed Central

Xie, Xi; Meesapyodsuk, Dauenpen

2017-01-01

ABSTRACT Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli. The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. coli fabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Thraustochytrium. IMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protist Thraustochytrium sp. strain ATCC 26185 and then expressing them in wild-type E. coli and mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes in E. coli. This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs. PMID:28213537

Biochemical analysis of the biosynthetic pathway of an anticancer tetracycline SF2575.

PubMed

Pickens, Lauren B; Kim, Woncheol; Wang, Peng; Zhou, Hui; Watanabe, Kenji; Gomi, Shuichi; Tang, Yi

2009-12-09

SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity toward a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with D-olivose and further acylation of the C4'-hydroxyl of D-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogues. In this study, we identified, sequenced, and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features, are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant-group addition is C-9 glycosylation, C-4 salicylation, and O-4' angelylcylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity toward substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group toward structure-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecarboxamide skeleton. Additional enzymes, such as cyclases C-6 methyltransferase and C-4/C-12a dihydroxylase, were functionally reconstituted.

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.

Biomarkers of Exposure to Toxic Substances. Volume 3: Proteomics, Biomarkers to Kidney and Organ Damage

DTIC Science & Technology

2009-05-01

not reveal any interesting proteins (Data is not shown). All of the spots turned out to be keratins. 3.1.2.2 Conclusions on puromycin toxicity...hamster) Odorant-binding protein precursor - Rattus norvegicus (Rat) 0.543 2.105 Oleandomycin polyketide synthase , modules 5...Using Government drawings, specifications, or other data included in this document for any purpose other than Government procurement does not

Graduate Training in Environmental and Marine Microbiology

DTIC Science & Technology

1999-05-31

metabolites biosynthesized by modular Type I polyketide synthases (PKS-I). We are investigating the possibility that "E. sertula" is the...completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information...the course of this grant. They have or soon will graduate ( one Masters and one Ph.D.) and have received awards based on their work. Due to these

Bioactivity Assessment of Indian Origin-Mangrove Actinobacteria against Candida albicans.

PubMed

Pavan Kumar, J G S; Gomathi, Ajitha; Gothandam, K M; Vasconcelos, Vitor

2018-02-12

Actinobacteria is found to have a potent metabolic activity against pathogens. The present study reveals the assessment of potent antifungal secondary metabolites from actinobacteria isolated from Indian marine mangrove sediments. The samples were collected from the coastal regions of Muthupet, Andaman and the Nicobar Islands. Identification was carried out using 16S rRNA analysis and biosynthetic genes (Polyketide synthase type I/II and Non-ribosomal peptide synthase) were screened. Actinobacteria were assayed for their antifungal activity against 16 clinical Candida albicans and the compound analysis was performed using gas chromatography-mass spectrometry GC-MS. The 31 actinobacterial strains were isolated and 16S rRNA gene sequencing revealed that this ecosystem is rich on actinobacteria, with Streptomyces as the predominant genus. The PCR based screening of biosynthetic genes revealed the presence of PKS-I in six strains, PKS-II in four strains and NRPS in 11 strains. The isolated actinobacteria VITGAP240 and VITGAP241 (two isolates) were found to have a potential antifungal activity against all the tested C. albicans . GC-MS results revealed that the actinobacterial compounds were belonging to heterocyclic, polyketides and peptides. Overall, the strains possess a wide spectrum of antifungal properties which affords the production of significant bioactive metabolites as potential antibiotics.

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.

Bioactivity Assessment of Indian Origin—Mangrove Actinobacteria against Candida albicans

PubMed Central

Pavan Kumar, J. G. S.; Gomathi, Ajitha; Vasconcelos, Vitor

2018-01-01

Actinobacteria is found to have a potent metabolic activity against pathogens. The present study reveals the assessment of potent antifungal secondary metabolites from actinobacteria isolated from Indian marine mangrove sediments. The samples were collected from the coastal regions of Muthupet, Andaman and the Nicobar Islands. Identification was carried out using 16S rRNA analysis and biosynthetic genes (Polyketide synthase type I/II and Non-ribosomal peptide synthase) were screened. Actinobacteria were assayed for their antifungal activity against 16 clinical Candida albicans and the compound analysis was performed using gas chromatography-mass spectrometry GC-MS. The 31 actinobacterial strains were isolated and 16S rRNA gene sequencing revealed that this ecosystem is rich on actinobacteria, with Streptomyces as the predominant genus. The PCR based screening of biosynthetic genes revealed the presence of PKS-I in six strains, PKS-II in four strains and NRPS in 11 strains. The isolated actinobacteria VITGAP240 and VITGAP241 (two isolates) were found to have a potential antifungal activity against all the tested C. albicans. GC-MS results revealed that the actinobacterial compounds were belonging to heterocyclic, polyketides and peptides. Overall, the strains possess a wide spectrum of antifungal properties which affords the production of significant bioactive metabolites as potential antibiotics. PMID:29439535

Identification and characterization of multiple curcumin synthases from the herb Curcuma longa.

PubMed

Katsuyama, Yohei; Kita, Tomoko; Horinouchi, Sueharu

2009-09-03

Curcuminoids are pharmaceutically important compounds isolated from the herb Curcuma longa. Two additional type III polyketide synthases, named CURS2 and CURS3, that are capable of curcuminoid synthesis were identified and characterized. In vitro analysis revealed that CURS2 preferred feruloyl-CoA as a starter substrate and CURS3 preferred both feruloyl-CoA and p-coumaroyl-CoA. These results suggested that CURS2 synthesizes curcumin or demethoxycurcumin and CURS3 synthesizes curcumin, bisdemethoxycurcumin and demethoxycurcumin. The availability of the substrates and the expression levels of the three different enzymes capable of curcuminoid synthesis with different substrate specificities might influence the composition of curcuminoids in the turmeric and in different cultivars.

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.

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

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

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.

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

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

Algorithms for Automated DNA Assembly

DTIC Science & Technology

2010-01-01

polyketide synthase gene cluster. Proc. Natl Acad. Sci. USA, 101, 15573–15578. 16. Shetty,R.P., Endy,D. and Knight,T.F. Jr (2008) Engineering BioBrick vectors...correct theoretical construction scheme is de- veloped manually, it is likely to be suboptimal by any number of cost metrics. Modular, robust and...to an exhaustive search on a small synthetic dataset and our results show that our algorithms can quickly find an optimal solution. Comparison with

Bacterium induces cryptic meroterpenoid pathway in the pathogenic fungus Aspergillus fumigatus.

PubMed

König, Claudia C; Scherlach, Kirstin; Schroeckh, Volker; Horn, Fabian; Nietzsche, Sandor; Brakhage, Axel A; Hertweck, Christian

2013-05-27

Stimulating encounter: The intimate, physical interaction between the soil-derived bacterium Streptomyces rapamycinicus and the human pathogenic fungus Aspergillus fumigatus led to the activation of an otherwise silent polyketide synthase (PKS) gene cluster coding for an unusual prenylated polyphenol (fumicycline A). The meroterpenoid pathway is regulated by a pathway-specific activator gene as well as by epigenetic factors. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

PubMed Central

Borejsza-Wysocki, W.; Hrazdina, G.

1996-01-01

p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response. PMID:12226219

Identification of candidate genes affecting Δ9-tetrahydrocannabinol biosynthesis in Cannabis sativa

PubMed Central

Marks, M. David; Tian, Li; Wenger, Jonathan P.; Omburo, Stephanie N.; Soto-Fuentes, Wilfredo; He, Ji; Gang, David R.; Weiblen, George D.; Dixon, Richard A.

2009-01-01

RNA isolated from the glands of a Δ9-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands. PMID:19581347

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.

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.

A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici.

PubMed

Zhang, Peng; Wang, Xiuna; Fan, Aili; Zheng, Yanjing; Liu, Xingzhong; Wang, Shihua; Zou, Huixi; Oakley, Berl R; Keller, Nancy P; Yin, Wen-Bing

2017-08-01

Spore pigmentation is very common in the fungal kingdom. The best studied pigment in fungi is melanin which coats the surface of single cell spores. What and how pigments function in a fungal species with multiple cell conidia is poorly understood. Here, we identified and deleted a polyketide synthase (PKS) gene PfmaE and showed that it is essential for multicellular conidial pigmentation and development in a plant endophytic fungus, Pestalotiopsis fici. To further characterize the melanin pathway, we utilized an advanced Aspergillus nidulans heterologous system for the expression of the PKS PfmaE and the Pfma gene cluster. By structural elucidation of the pathway metabolite scytalone in A. nidulans, we provided chemical evidence that the Pfma cluster synthesizes DHN melanin. Combining genetic deletion and combinatorial gene expression of Pfma cluster genes, we determined that the putative reductase PfmaG and the PKS are sufficient for the synthesis of scytalone. Feeding scytalone back to the P. fici ΔPfmaE mutant restored pigmentation and multicellular adherence of the conidia. These results cement a growing understanding that pigments are essential not simply for protection of spores from biotic and abiotic stresses but also for spore structural development. © 2017 John Wiley & Sons Ltd.

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.

De Novo Sequencing of Hypericum perforatum Transcriptome to Identify Potential Genes Involved in the Biosynthesis of Active Metabolites

PubMed Central

He, Miao; Wang, Ying; Hua, Wenping; Zhang, Yuan; Wang, Zhezhi

2012-01-01

Background Hypericum perforatum L. (St. John’s wort) is a medicinal plant with pharmacological properties that are antidepressant, anti-inflammatory, antiviral, anti-cancer, and antibacterial. Its major active metabolites are hypericins, hyperforins, and melatonin. However, little genetic information is available for this species, especially that concerning the biosynthetic pathways for active ingredients. Methodology/Principal Findings Using de novo transcriptome analysis, we obtained 59,184 unigenes covering the entire life cycle of these plants. In all, 40,813 unigenes (68.86%) were annotated and 2,359 were assigned to secondary metabolic pathways. Among them, 260 unigenes are involved in the production of hypericin, hyperforin, and melatonin. Another 2,291 unigenes are classified as potential Type III polyketide synthase. Our BlastX search against the AGRIS database reveals 1,772 unigenes that are homologous to 47 known Arabidopsis transcription factor families. Further analysis shows that 10.61% (6,277) of these unigenes contain 7,643 SSRs. Conclusion We have identified a set of putative genes involved in several secondary metabolism pathways, especially those related to the synthesis of its active ingredients. Our results will serve as an important platform for public information about gene expression, genomics, and functional genomics in H. perforatum. PMID:22860059

Biosynthetic Potential of Bioactive Streptomycetes Isolated From Arid Region of the Thar Desert, Rajasthan (India)

PubMed Central

Masand, Meeta; Sivakala, Kunjukrishnan Kamalakshi; Menghani, Ekta; Thinesh, Thangathurai; Anandham, Rangasamy; Sharma, Gaurav; Sivakumar, Natesan; Jebakumar, Solomon R. D.; Jose, Polpass Arul

2018-01-01

Acquisition of Actinobacteria, especially Streptomyces from previously underexplored habitats and the exploration of their biosynthetic potential have gained much attention in the rejuvenated antibiotics search programs. Herein, we isolated some Streptomyces strains, from an arid region of the Great Indian Thar Desert, which possess an ability to produce novel bioactive compounds. Twenty-one morphologically distinctive strains differing in their aerial and substrate mycelium were isolated by employing a stamping method. Among them, 12 strains were identified by a two-level antimicrobial screening method, exerting antimicrobial effects against a panel of indicator strains including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus species. Based on their potent antimicrobial activity, four isolates were further explored by 16S rRNA gene-based identification, genetic screening, and metabolomic analysis; and it was found that these strains belong to the genus Streptomyces. The selected strains were found to have polyketide synthase and non-ribosomal peptide synthetase systems. In addition, extracellular metabolomic screening revealed that the isolates produced analogs of doxorubicinol, pyrromycin, erythromycin, and 6-13 other putative novel metabolites. These results demonstrate the significance of Streptomyces inhabiting the arid region of Thar Desert, suggesting that similar arid environments can be considered as the reservoirs of novel Streptomyces strains that could have biotechnological significance. PMID:29720968

A Systems Biology Framework for Modeling Metabolic Enzyme Inhibition of Mycobacterium Tuberculosis

DTIC Science & Technology

2009-09-15

Quadri LE: Assembly of aryl-capped siderophores by modular peptide synthetases and polyketide synthases . Mol Microbiol 2000, 37:1-12. 51. Chou CJ...opportunities for therapeutic intervention. Results: We developed a mathematical framework to simulate the effects on the growth of a pathogen when enzymes in... on the growth of M. tuberculosis in a medium whose carbon source was restricted to fatty acids, and that of the 5’-O-(N-salicylsulfamoyl) adenosine

European Science Notes Information Bulletin Reports on Current European/ Middle Eastern Science

DTIC Science & Technology

1989-05-01

different polyketide synthases have varying 13 ESNIB 89-05 degrees of conserved DNA sequence homology so that Protein Design genes for one antibiotic (for...information on this myosin isoform of any organism. Ex-wthwo 2kAandtolkaliorregulatory light chains (16 to21pression of this novel myosin was studied...expected with any new material. The industrial com- an accurate displacement. One of these was by L. Kiese- munity with its tremendous market is probably

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.

Biosynthesis of the antifungal haterumalide, oocydin A, in Serratia, and its regulation by quorum sensing, RpoS and Hfq

PubMed Central

Matilla, Miguel A; Leeper, Finian J; Salmond, George P C

2015-01-01

Polyketides represent an important class of bioactive natural products with a broad range of biological activities. We identified recently a large trans-acyltransferase (AT) polyketide synthase gene cluster responsible for the biosynthesis of the antifungal, anti-oomycete and antitumor haterumalide, oocydin A (ooc). Using genome sequencing and comparative genomics, we show that the ooc gene cluster is widespread within biocontrol and phytopathogenic strains of the enterobacteria, Serratia and Dickeya. The analysis of in frame deletion mutants confirmed the role of a hydroxymethylglutaryl-coenzyme A synthase cassette, three flavin-dependent tailoring enzymes, a free-standing acyl carrier protein and two hypothetical proteins in oocydin A biosynthesis. The requirement of the three trans-acting AT domains for the biosynthesis of the macrolide was also demonstrated. Expression of the ooc gene cluster was shown to be positively regulated by an N-acyl-L-homoserine lactone-based quorum sensing system, but operating in a strain-dependent manner. At a post-transcriptional level, the RNA chaperone, Hfq, plays a key role in oocydin A biosynthesis. The Hfq-dependent regulation is partially mediated by the stationary phase sigma factor, RpoS, which was also shown to positively regulate the synthesis of the macrolide. Our results reveal differential regulation of the divergently transcribed ooc transcriptional units, highlighting the complexity of oocydin A production. PMID:25753587

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.

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

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

Draft genome sequence of a human-associated isolate of Haloferax alexandrinus strain Arc-hr, an extremely halophilic archaea.

PubMed

Khelaifia, S; Caputo, A; Djossou, F; Raoult, D

2017-01-01

We report the draft genome sequence of Haloferax alexandrinus strain Arc-hr (CSUR P798), isolated from the human gut of a 10-year-old Amazonian individual. Its 3 893 626 bp genome exhibits a 66.00% GC content. The genome of the strain Arc-hr contains 37 genes identified as ORFans, seven genes associated to halocin and 11 genes associated with polyketide synthases or nonribosomal peptide synthetases.

Terminal Olefin Profiles and Phylogenetic Analyses of Olefin Synthases of Diverse Cyanobacterial Species.

PubMed

Zhu, Tao; Scalvenzi, Thibault; Sassoon, Nathalie; Lu, Xuefeng; Gugger, Muriel

2018-07-01

Cyanobacteria can synthesize alkanes and alkenes, which are considered to be infrastructure-compatible biofuels. In terms of physiological function, cyanobacterial hydrocarbons are thought to be essential for membrane flexibility for cell division, size, and growth. The genetic basis for the biosynthesis of terminal olefins (1-alkenes) is a modular type I polyketide synthase (PKS) termed olefin synthase (Ols). The modular architectures of Ols and structural characteristics of alkenes have been investigated only in a few species of the small percentage (approximately 10%) of cyanobacteria that harbor putative Ols pathways. In this study, investigations of the domains, modular architectures, and phylogenies of Ols in 28 cyanobacterial strains suggested distinctive pathway evolution. Structural feature analyses revealed 1-alkenes with three carbon chain lengths (C 15 , C 17 , and C 19 ). In addition, the total cellular fatty acid profile revealed the diversity of the carbon chain lengths, while the fatty acid feeding assay indicated substrate carbon chain length specificity of cyanobacterial Ols enzymes. Finally, in silico analyses suggested that the N terminus of the modular Ols enzyme exhibited characteristics typical of a fatty acyl-adenylate ligase (FAAL), suggesting a mechanism of fatty acid activation via the formation of acyl-adenylates. Our results shed new light on the diversity of cyanobacterial terminal olefins and a mechanism for substrate activation in the biosynthesis of these olefins. IMPORTANCE Cyanobacterial terminal olefins are hydrocarbons with promising applications as advanced biofuels. Despite the basic understanding of the genetic basis of olefin biosynthesis, the structural diversity and phylogeny of the key modular olefin synthase (Ols) have been poorly explored. An overview of the chemical structural traits of terminal olefins in cyanobacteria is provided in this study. In addition, we demonstrated by in vivo fatty acid feeding assays that cyanobacterial Ols enzymes might exhibit substrate carbon chain length specificity. Furthermore, by performing bioinformatic analyses, we observed that the substrate activation domain of Ols exhibited features typical of a fatty acyl-adenylate ligase (FAAL), which activates fatty acids by converting them to fatty acyl-adenylates. Our results provide further insight into the chemical structures of terminal olefins and further elucidate the mechanism of substrate activation for terminal olefin biosynthesis in cyanobacteria. Copyright © 2018 American Society for Microbiology.

Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta.

PubMed

Zhu, Yu-Cheng; Specht, Charles A; Dittmer, Neal T; Muthukrishnan, Subbaratnam; Kanost, Michael R; Kramer, Karl J

2002-11-01

Glycosyltransferases are enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. One type of glycosyltransferase is chitin synthase, a very important enzyme in biology, which is utilized by insects, fungi, and other invertebrates to produce chitin, a polysaccharide of beta-1,4-linked N-acetylglucosamine. Chitin is an important component of the insect's exoskeletal cuticle and gut lining. To identify and characterize a chitin synthase gene of the tobacco hornworm, Manduca sexta, degenerate primers were designed from two highly conserved regions in fungal and nematode chitin synthase protein sequences and then used to amplify a similar region from Manduca cDNA. A full-length cDNA of 5152 nucleotides was assembled for the putative Manduca chitin synthase gene, MsCHS1, and sequencing of genomic DNA verified the contiguity of the sequence. The MsCHS1 cDNA has an ORF of 4692 nucleotides that encodes a transmembrane protein of 1564 amino acid residues with a mass of approximately 179 kDa (GenBank no. AY062175). It is most similar, over its entire length of protein sequence, to putative chitin synthases from other insects and nematodes, with 68% identity to enzymes from both the blow fly, Lucilia cuprina, and the fruit fly, Drosophila melanogaster. The similarity with fungal chitin synthases is restricted to the putative catalytic domain, and the MsCHS1 protein has, at equivalent positions, several amino acids that are essential for activity as revealed by mutagenesis of the fungal enzymes. A 5.3-kb transcript of MsCHS1 was identified by northern blot hybridization of RNA from larval epidermis, suggesting that the enzyme functions to make chitin deposited in the cuticle. Further examination by RT-PCR showed that MsCHS1 expression is regulated in the epidermis, with the amount of transcript increasing during phases of cuticle deposition.

Identification of the first diphenyl ether gene cluster for pestheic acid biosynthesis in plant endophyte Pestalotiopsis fici.

PubMed

Xu, Xinxin; Liu, Ling; Zhang, Fan; Wang, Wenzhao; Li, Jinyang; Guo, Liangdong; Che, Yongsheng; Liu, Gang

2014-01-24

The diphenyl ether pestheic acid was isolated from the endophytic fungus Pestalotiopsis fici, which is proposed to be the biosynthetic precursor of the unique chloropupukeananes. The pestheic acid biosynthetic gene (pta) cluster was identified in the fungus through genome scanning. Sequence analysis revealed that this gene cluster encodes a nonreducing polyketide synthase, a number of modification enzymes, and three regulators. Gene disruption and intermediate analysis demonstrated that the biosynthesis proceeded through formation of the polyketide backbone, cyclization of a polyketo acid to a benzophenone, chlorination, and formation of the diphenyl ether skeleton through oxidation and hydrolyzation. A dihydrogeodin oxidase gene, ptaE, was essential for diphenyl ether formation, and ptaM encoded a flavin-dependent halogenase catalyzing chlorination in the biosynthesis. Identification of the pta cluster laid the foundation to decipher the genetic and biochemical mechanisms involved in the pathway. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Oxidative dearomatisation: the key step of sorbicillinoid biosynthesis† †Electronic supplementary information (ESI) available: Containing all experimental details. See DOI: 10.1039/c3sc52911h Click here for additional data file.

PubMed Central

Fahad, Ahmed al; Abood, Amira; Fisch, Katja M.; Osipow, Anna; Davison, Jack; Avramović, Marija; Butts, Craig P.; Piel, Jörn; Simpson, Thomas J.

2014-01-01

An FAD-dependent monooxygenase encoding gene (SorbC) was cloned from Penicillium chrysogenum E01-10/3 and expressed as a soluble protein in Escherichia coli. The enzyme efficiently performed the oxidative dearomatisation of sorbicillin and dihydrosorbicillin to give sorbicillinol and dihydrosorbicillinol respectively. Bioinformatic examination of the gene cluster surrounding SorbC indicated the presence of two polyketide synthase (PKS) encoding genes designated sorbA and sorbB. The gene sorbA-encodes a highly reducing iterative PKS while SorbB encodes a non-reducing iterative PKS which features a reductive release domain usually involved in the production of polyketide aldehydes. Using these observations and previously reported results from isotopic feeding experiments a new and simpler biosynthetic route to the sorbicillin class of secondary metabolites is proposed which is consistent with all reported experimental results. PMID:25580210

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

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.

Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

PubMed

Zheng, Desen; Burr, Thomas J

2016-02-01

Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

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.

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.

Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao.

PubMed

Mares, Joise Hander; Gramacho, Karina Peres; Santos, Everton Cruz; da Silva Santiago, André; Santana, Juliano Oliveira; de Sousa, Aurizângela Oliveira; Alvim, Fátima Cerqueira; Pirovani, Carlos Priminho

2017-08-17

Moniliophthora perniciosa is a phytopathogenic fungus responsible for witches' broom disease of cacao trees (Theobroma cacao L.). Understanding the molecular events during germination of the pathogen may enable the development of strategies for disease control in these economically important plants. In this study, we determined a comparative proteomic profile of M. perniciosa basidiospores during germination by two-dimensional SDS-PAGE and mass spectrometry. A total of 316 proteins were identified. Molecular changes during the development of the germinative tube were identified by a hierarchical clustering analysis based on the differential accumulation of proteins. Proteins associated with fungal filamentation, such as septin and kinesin, were detected only 4 h after germination (hag). A transcription factor related to biosynthesis of the secondary metabolite fumagillin, which can form hybrids with polyketides, was induced 2 hag, and polyketide synthase was observed 4 hag. The accumulation of ATP synthase, binding immunoglobulin protein (BiP), and catalase was validated by western blotting. In this study, we showed variations in protein expression during the early germination stages of fungus M. perniciosa. Proteins associated with fungal filamentation, and consequently with virulence, were detected in basidiospores 4 hag., for example, septin and kinesin. We discuss these results and propose a model of the germination of fungus M. perniciosa. This research can help elucidate the mechanisms underlying basic processes of host invasion and to develop strategies for control of the disease.

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.

Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

PubMed Central

Upadhyay, Srijana; Xu, Xinping

2016-01-01

ABSTRACT Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism. PMID:27879337

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

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

Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages.

PubMed

Shulse, Christine N; Allen, Eric E

2011-01-01

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as "Pfa synthases". In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of "secondary lipids" to describe these biosynthetic pathways and products, a proposition consistent with the "secondary metabolite" vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages.

Genomics reveals traces of fungal phenylpropanoid-flavonoid metabolic pathway in the f ilamentous fungus Aspergillus oryzae.

PubMed

Juvvadi, Praveen Rao; Seshime, Yasuyo; Kitamoto, Katsuhiko

2005-12-01

Fungal secondary metabolites constitute a wide variety of compounds which either play a vital role in agricultural, pharmaceutical and industrial contexts, or have devastating effects on agriculture, animal and human affairs by virtue of their toxigenicity. Owing to their beneficial and deleterious characteristics, these complex compounds and the genes responsible for their synthesis have been the subjects of extensive investigation by microbiologists and pharmacologists. A majority of the fungal secondary metabolic genes are classified as type I polyketide synthases (PKS) which are often clustered with other secondary metabolism related genes. In this review we discuss on the significance of our recent discovery of chalcone synthase (CHS) genes belonging to the type III PKS superfamily in an industrially important fungus, Aspergillus oryzae. CHS genes are known to play a vital role in the biosynthesis of flavonoids in plants. A comparative genome analyses revealed the unique character of A. oryzae with four CHS-like genes (csyA, csyB, csyC and csyD) amongst other Aspergilli (Aspergillus nidulans and Aspergillus fumigatus) which contained none of the CHS-like genes. Some other fungi such as Neurospora crassa, Fusarium graminearum, Magnaporthe grisea, Podospora anserina and Phanerochaete chrysosporium also contained putative type III PKSs, with a phylogenic distinction from bacteria and plants. The enzymatically active nature of these newly discovered homologues is expected owing to the conservation in the catalytic residues across the different species of plants and fungi, and also by the fact that a majority of these genes (csyA, csyB and csyD) were expressed in A. oryzae. While this finding brings filamentous fungi closer to plants and bacteria which until recently were the only ones considered to possess the type III PKSs, the presence of putative genes encoding other principal enzymes involved in the phenylpropanoid and flavonoid biosynthesis (viz., phenylalanine ammonia-lyase, cinnamic acid hydroxylase and p-coumarate CoA ligase) in the A. oryzae genome undoubtedly prove the extent of its metabolic diversity. Since many of these genes have not been identified earlier, knowledge on their corresponding products or activities remain undeciphered. In future, it is anticipated that these enzymes may be reasonable targets for metabolic engineering in fungi to produce agriculturally and nutritionally important metabolites.

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.

Single Cell Genome Amplification Accelerates Identification of the Apratoxin Biosynthetic Pathway from a Complex Microbial Assemblage

PubMed Central

Grindberg, Rashel V.; Ishoey, Thomas; Brinza, Dumitru; Esquenazi, Eduardo; Coates, R. Cameron; Liu, Wei-ting; Gerwick, Lena; Dorrestein, Pieter C.; Pevzner, Pavel; Lasken, Roger; Gerwick, William H.

2011-01-01

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites. PMID:21533272

Regulation of Fumonisin B1 Biosynthesis and Conidiation in Fusarium verticillioides by a Cyclin-Like (C-Type) Gene, FCC1†

PubMed Central

Shim, Won-Bo; Woloshuk, Charles P.

2001-01-01

Fumonisins are a group of mycotoxins produced in corn kernels by the plant-pathogenic fungus Fusarium verticillioides. A mutant of the fungus, FT536, carrying a disrupted gene named FCC1 (for Fusarium cyclin C1) resulting in altered fumonisin B1 biosynthesis was generated. FCC1 contains an open reading frame of 1,018 bp, with one intron, and encodes a putative 319-amino-acid polypeptide. This protein is similar to UME3 (also called SRB11 or SSN8), a cyclin C of Saccharomyces cerevisiae, and contains three conserved motifs: a cyclin box, a PEST-rich region, and a destruction box. Also similar to the case for C-type cyclins, FCC1 was constitutively expressed during growth. When strain FT536 was grown on corn kernels or on defined minimal medium at pH 6, conidiation was reduced and FUM5, the polyketide synthase gene involved in fumonisin B1 biosynthesis, was not expressed. However, when the mutant was grown on a defined minimal medium at pH 3, conidiation was restored, and the blocks in expression of FUM5 and fumonisin B1 production were suppressed. Our data suggest that FCC1 plays an important role in signal transduction regulating secondary metabolism (fumonisin biosynthesis) and fungal development (conidiation) in F. verticillioides. PMID:11282612

Mycobacterium ahvazicum sp. nov., the nineteenth species of the Mycobacterium simiae complex.

PubMed

Bouam, Amar; Heidarieh, Parvin; Shahraki, Abodolrazagh Hashemi; Pourahmad, Fazel; Mirsaeidi, Mehdi; Hashemzadeh, Mohamad; Baptiste, Emeline; Armstrong, Nicholas; Levasseur, Anthony; Robert, Catherine; Drancourt, Michel

2018-03-07

Four slowly growing mycobacteria isolates were isolated from the respiratory tract and soft tissue biopsies collected in four unrelated patients in Iran. Conventional phenotypic tests indicated that these four isolates were identical to Mycobacterium lentiflavum while 16S rRNA gene sequencing yielded a unique sequence separated from that of M. lentiflavum. One representative strain AFP-003 T was characterized as comprising a 6,121,237-bp chromosome (66.24% guanosine-cytosine content) encoding for 5,758 protein-coding genes, 50 tRNA and one complete rRNA operon. A total of 2,876 proteins were found to be associated with the mobilome, including 195 phage proteins. A total of 1,235 proteins were found to be associated with virulence and 96 with toxin/antitoxin systems. The genome of AFP-003 T has the genetic potential to produce secondary metabolites, with 39 genes found to be associated with polyketide synthases and non-ribosomal peptide syntases and 11 genes encoding for bacteriocins. Two regions encoding putative prophages and three OriC regions separated by the dnaA gene were predicted. Strain AFP-003 T genome exhibits 86% average nucleotide identity with Mycobacterium genavense genome. Genetic and genomic data indicate that strain AFP-003 T is representative of a novel Mycobacterium species that we named Mycobacterium ahvazicum, the nineteenth species of the expanding Mycobacterium simiae complex.

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.

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

Insights into the Biosynthesis of 12-Membered Resorcylic Acid Lactones from Heterologous Production in Saccharomyces cerevisiae

PubMed Central

2015-01-01

The phytotoxic fungal polyketides lasiodiplodin and resorcylide inhibit human blood coagulation factor XIIIa, mineralocorticoid receptors, and prostaglandin biosynthesis. These secondary metabolites belong to the 12-membered resorcylic acid lactone (RAL12) subclass of the benzenediol lactone (BDL) family. Identification of genomic loci for the biosynthesis of lasiodiplodin from Lasiodiplodia theobromae and resorcylide from Acremonium zeae revealed collaborating iterative polyketide synthase (iPKS) pairs whose efficient heterologous expression in Saccharomyces cerevisiae provided a convenient access to the RAL12 scaffolds desmethyl-lasiodiplodin and trans-resorcylide, respectively. Lasiodiplodin production was reconstituted in the heterologous host by co-expressing an O-methyltransferase also encoded in the lasiodiplodin cluster, while a glutathione-S-transferase was found not to be necessary for heterologous production. Clarification of the biogenesis of known resorcylide congeners in the heterologous host helped to disentangle the roles that biosynthetic irregularities and chemical interconversions play in generating chemical diversity. Observation of 14-membered RAL homologues during in vivo heterologous biosynthesis of RAL12 metabolites revealed “stuttering” by fungal iPKSs. The close global and domain-level sequence similarities of the orthologous BDL synthases across different structural subclasses implicate repeated horizontal gene transfers and/or cluster losses in different fungal lineages. The absence of straightforward correlations between enzyme sequences and product structural features (the size of the macrocycle, the conformation of the exocyclic methyl group, or the extent of reduction by the hrPKS) suggest that BDL structural variety is the result of a select few mutations in key active site cavity positions. PMID:24597618

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

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

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

Large-Scale Phylogenetic Classification of Fungal Chitin Synthases and Identification of a Putative Cell-Wall Metabolism Gene Cluster in Aspergillus Genomes

PubMed Central

Pacheco-Arjona, Jose Ramon; Ramirez-Prado, Jorge Humberto

2014-01-01

The cell wall is a protective and versatile structure distributed in all fungi. The component responsible for its rigidity is chitin, a product of chitin synthase (Chsp) enzymes. There are seven classes of chitin synthase genes (CHS) and the amount and type encoded in fungal genomes varies considerably from one species to another. Previous Chsp sequence analyses focused on their study as individual units, regardless of genomic context. The identification of blocks of conserved genes between genomes can provide important clues about the interactions and localization of chitin synthases. On the present study, we carried out an in silico search of all putative Chsp encoded in 54 full fungal genomes, encompassing 21 orders from five phyla. Phylogenetic studies of these Chsp were able to confidently classify 347 out of the 369 Chsp identified (94%). Patterns in the distribution of Chsp related to taxonomy were identified, the most prominent being related to the type of fungal growth. More importantly, a synteny analysis for genomic blocks centered on class IV Chsp (the most abundant and widely distributed Chsp class) identified a putative cell wall metabolism gene cluster in members of the genus Aspergillus, the first such association reported for any fungal genome. PMID:25148134

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.

Characterization of three chalcone synthase-like genes from apple (Malus x domestica Borkh.).

PubMed

Yahyaa, Mosaab; Ali, Samah; Davidovich-Rikanati, Rachel; Ibdah, Muhammad; Shachtier, Alona; Eyal, Yoram; Lewinsohn, Efraim; Ibdah, Mwafaq

2017-08-01

Apple (Malus x domestica Brokh.) is a widely cultivated deciduous tree species of significant economic importance. Apple leaves accumulate high levels of flavonoids and dihydrochalcones, and their formation is dependent on enzymes of the chalcone synthase family. Three CHS genes were cloned from apple leaves and expressed in Escherichia coli. The encoded recombinant enzymes were purified and functionally characterized. In-vitro activity assays indicated that MdCHS1, MdCHS2 and MdCHS3 code for proteins exhibiting polyketide synthase activity that accepted either p-dihydrocoumaroyl-CoA, p-coumaroyl-CoA, or cinnamoyl-CoA as starter CoA substrates in the presence of malonyl-CoA, leading to production of phloretin, naringenin chalcone, and pinocembrin chalcone. MdCHS3 coded a chalcone-dihydrochalcone synthase enzyme with narrower substrate specificity than the previous ones. The apparent Km values of MdCHS3 for p-dihydrocoumaryl-CoA and p-coumaryl-CoA were both 5.0 μM. Expression analyses of MdCHS genes varied according to tissue type. MdCHS1, MdCHS2 and MdCHS3 expression levels were associated with the levels of phloretin accumulate in the respective tissues. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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.

Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

PubMed

Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

2015-03-01

The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in localized combination of conserved residues.

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.

Proteomics analysis of Fusarium proliferatum under various initial pH during fumonisin production.

PubMed

Li, Taotao; Gong, Liang; Wang, Yong; Chen, Feng; Gupta, Vijai Kumar; Jian, Qijie; Duan, Xuewu; Jiang, Yueming

2017-07-05

Fusarium proliferatum as a fungal pathogen can produce fumonisin which causes a great threat to animal and human health. Proteomic approach was a useful tool for investigation into mycotoxin biosynthesis in fungal pathogens. In this study, we analyzed the fumonisin content and mycelium proteins of Fusarium proliferatum cultivated under the initial pH5 and 10. Fumonisin production after 10days was significantly induced in culture condition at pH10 than pH5. Ninety nine significantly differently accumulated protein spots under the two pH conditions were detected using two dimensional polyacrylamide gel electrophoresis and 89 of these proteins were successfully identified by MALDI-TOF/TOF and LC-ESI-MS/MS analysis. Among these 89 proteins, 45 were up-regulated at pH10 while 44 were up-accumulated at pH5. At pH10, these proteins were found to involve in the modification of fumonisin backbone including up-regulated polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase, which might contribute to the induction of fumonisin production. At pH5, these up-regulated proteins such as l-amino-acid oxidase, isocitrate dehydrogenase and citrate lyase might inhibit the condensation of fumonisin backbone, resulting in reduced production of fumonisins. These results may help us to understand the molecular mechanism of the fumonisin synthesis in F. proliferatum. To extend our understanding of the mechanism of the fumonisin biosynthesis of F. proliferatum, we reported the fumonisin production in relation to the differential proteins of F. proliferatum mycelium under two pH culture conditions. Among these 89 identified spots, 45 were up-accumulated at pH10 while 44 were up-accumulated at pH5. Our results revealed that increased fumonisin production at pH10 might be related to the induction of fumonisin biosynthesis caused by up-regulation of polyketide synthase, cytochrome P450, S-adenosylmethionine synthase and O-methyltransferase. Meanwhile, the up-regulation of l-amino-acid oxidase, isocitrate dehydrogenase and citrate lyase at pH5 might be related to the inhibition of the condensation of fumonisin backbone, resulting in reduced production of fumonisin. These results may help us to understand better the molecular mechanism of the fumonisin synthesis in F. proliferatum and then broaden the current knowledge of the mechanism of the fumonisin biosynthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Cellulose in Cyanobacteria. Origin of Vascular Plant Cellulose Synthase?

PubMed Central

Nobles, David R.; Romanovicz, Dwight K.; Brown, R. Malcolm

2001-01-01

Although cellulose biosynthesis among the cyanobacteria has been suggested previously, we present the first conclusive evidence, to our knowledge, of the presence of cellulose in these organisms. Based on the results of x-ray diffraction, electron microscopy of microfibrils, and cellobiohydrolase I-gold labeling, we report the occurrence of cellulose biosynthesis in nine species representing three of the five sections of cyanobacteria. Sequence analysis of the genomes of four cyanobacteria revealed the presence of multiple amino acid sequences bearing the DDD35QXXRW motif conserved in all cellulose synthases. Pairwise alignments demonstrated that CesAs from plants were more similar to putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and Nostoc punctiforme American Type Culture Collection 29133 than any other cellulose synthases in the database. Multiple alignments of putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and N. punctiforme American Type Culture Collection 29133 with the cellulose synthases of other prokaryotes, Arabidopsis, Gossypium hirsutum, Populus alba × Populus tremula, corn (Zea mays), and Dictyostelium discoideum showed that cyanobacteria share an insertion between conserved regions U1 and U2 found previously only in eukaryotic sequences. Furthermore, phylogenetic analysis indicates that the cyanobacterial cellulose synthases share a common branch with CesAs of vascular plants in a manner similar to the relationship observed with cyanobacterial and chloroplast 16s rRNAs, implying endosymbiotic transfer of CesA from cyanobacteria to plants and an ancient origin for cellulose synthase in eukaryotes. PMID:11598227

PCR screening reveals considerable unexploited biosynthetic potential of ansamycins and a mysterious family of AHBA-containing natural products in actinomycetes.

PubMed

Wang, H-X; Chen, Y-Y; Ge, L; Fang, T-T; Meng, J; Liu, Z; Fang, X-Y; Ni, S; Lin, C; Wu, Y-Y; Wang, M-L; Shi, N-N; He, H-G; Hong, K; Shen, Y-M

2013-07-01

Ansamycins are a family of macrolactams that are synthesized by type I polyketide synthase (PKS) using 3-amino-5-hydroxybenzoic acid (AHBA) as the starter unit. Most members of the family have strong antimicrobial, antifungal, anticancer and/or antiviral activities. We aimed to discover new ansamycins and/or other AHBA-containing natural products from actinobacteria. Through PCR screening of AHBA synthase gene, we identified 26 AHBA synthase gene-positive strains from 206 plant-associated actinomycetes (five positives) and 688 marine-derived actinomycetes (21 positives), representing a positive ratio of 2·4-3·1%. Twenty-five ansamycins, including eight new compounds, were isolated from six AHBA synthase gene-positive strains through TLC-guided fractionations followed by repeated column chromatography. To gain information about those potential ansamycin gene clusters whose products were unknown, seven strains with phylogenetically divergent AHBA synthase genes were subjected to fosmid library construction. Of the seven gene clusters we obtained, three show characteristics for typical ansamycin gene clusters, and other four, from Micromonospora spp., appear to lack the amide synthase gene, which is unusual for ansamycin biosynthesis. The gene composition of these four gene clusters suggests that they are involved in the biosynthesis of a new family of hybrid PK-NRP compounds containing AHBA substructure. PCR screening of AHBA synthase is an efficient approach to discover novel ansamycins and other AHBA-containing natural products. This work demonstrates that the AHBA-based screening method is a useful approach for discovering novel ansamycins and other AHBA-containing natural products from new microbial resources. Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.

Biosynthetic Potential of Phylogenetically Unique Endophytic Actinomycetes from Tropical Plants▿ †

PubMed Central

Janso, Jeffrey E.; Carter, Guy T.

2010-01-01

The culturable diversity of endophytic actinomycetes associated with tropical, native plants is essentially unexplored. In this study, 123 endophytic actinomycetes were isolated from tropical plants collected from several locations in Papua New Guinea and Mborokua Island, Solomon Islands. Isolates were found to be prevalent in roots but uncommon in leaves. Initially, isolates were dereplicated to the strain level by ribotyping. Subsequent characterization of 105 unique strains by 16S rRNA gene sequence analysis revealed that 17 different genera were represented, and rare genera, such as Sphaerisporangium and Planotetraspora, which have never been previously reported to be endophytic, were quite prevalent. Phylogenetic analyses grouped many of the strains into clades distinct from known genera within Thermomonosporaceae and Micromonosporaceae, indicating that they may be unique genera. Bioactivity testing and liquid chromatography-mass spectrometry (LC-MS) profiling of crude fermentation extracts were performed on 91 strains. About 60% of the extracts exhibited bioactivity or displayed LC-MS profiles with spectra indicative of secondary metabolites. The biosynthetic potential of 29 nonproductive strains was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. Despite their lack of detectable secondary metabolite production in fermentation, most were positive for type I (66%) and type II (79%) PKS genes, and all were positive for NRPS genes. These results suggest that tropical plants from New Guinea and the adjacent archipelago are hosts to unique endophytic actinomycetes that possess significant biosynthetic potential. PMID:20472734

Gatekeeping versus Promiscuity in the Early Stages of the Andrimid Biosynthetic Assembly Line

PubMed Central

Magarvey, Nathan A.; Fortin, Pascal D.; Thomas, Paul M.; Kelleher, Neil L.; Walsh, Christopher T.

2009-01-01

The antibiotic andrimid, a nanomolar inhibitor of bacterial acetyl coenzyme A carboxylase, is generated on an unusual polyketide/nonribosomal pep-tide enzyme assembly line in that all thiolation (T) domains/small-molecule building stations are on separate proteins. In addition, a transglutaminase homologue is used to condense andrimid building blocks together on the andrimid assembly line. The first two modules of the andrimid assembly line yields an octatrienoyl-β-Phe-thioester tethered to the AdmI T domain, with amide bond formation carried out by a free-standing transglutaminase homologue AdmF. Analysis of the aminomutase AdmH reveals its specific conversion from l-Phe to (S)-β-Phe, which in turn is activated by AdmJ and ATP to form (S)-β-Phe-aminoacyl-AMP. AdmJ then transfers the (S)-β-Phe moiety to one of the free-standing T domains, AdmI, but not AdmA, which instead gets loaded with an octatrienoyl group by other enzymes. AdmF, the amide synthase, will accept a variety of acyl groups in place of the octatrienoyl donor if presented on either AdmA or AdmI. AdmF will also use either stereoisomer of phenylalanine or β-Phe when presented on AdmA and AdmI, but not when placed on noncognate T domains. Further, we show the polyketide synthase proteins responsible for the polyunsaturated acyl cap can be bypassed in vitro with N-acetylcysteamine as a low-molecular-weight acyl donor to AdmF and also in vivo in an Escherichia coli strain bearing the andrimid biosynthetic gene cluster with a knockout in admA. PMID:18652473

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

Molecular dynamics simulation and binding free energy studies of novel leads belonging to the benzofuran class inhibitors of Mycobacterium tuberculosis Polyketide Synthase 13.

PubMed

Cruz, Jorddy N; Costa, José F S; Khayat, André S; Kuca, Kamil; Barros, Carlos A L; Neto, A M J C

2018-05-04

In this work, the binding mechanism of new Polyketide Synthase 13 (Pks13) inhibitors has been studied through molecular dynamics simulation and free energy calculations. The drug Tam1 and its analogs, belonging to the benzofuran class, were submitted to 100 ns simulations, and according to the results obtained for root mean square deviation, all the simulations converged from approximately 30 ns. For the analysis of backbone flotation, the root mean square fluctuations were plotted for the Cα atoms; analysis revealed that the greatest fluctuation occurred in the residues that are part of the protein lid domain. The binding free energy value (ΔG bind ) obtained for the Tam16 lead molecule was of -51.43 kcal/mol. When comparing this result with the ΔG bind values for the remaining analogs, the drug Tam16 was found to be the highest ranked: this result is in agreement with the experimental results obtained by Aggarwal and collaborators, where it was verified that the IC 50 for Tam16 is the smallest necessary to inhibit the Pks13 (IC 50  = 0.19 μM). The energy decomposition analysis suggested that the residues which most interact with inhibitors are: Ser1636, Tyr1637, Asn1640, Ala1667, Phe1670, and Tyr1674, from which the greatest energy contribution to Phe1670 was particularly notable. For the lead molecule Tam16, a hydrogen bond with the hydroxyl of the phenol not observed in the other analogs induced a more stable molecular structure. Aggarwal and colleagues reported this hydrogen bonding as being responsible for the stability of the molecule, optimizing its physic-chemical, toxicological, and pharmacokinetic properties.

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

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.

Linking secondary metabolites to biosynthesis genes in the fungal endophyte Cyanodermella asteris: The anti-cancer bisanthraquinone skyrin.

PubMed

Jahn, Linda; Schafhauser, Thomas; Wibberg, Daniel; Rückert, Christian; Winkler, Anika; Kulik, Andreas; Weber, Tilmann; Flor, Liane; van Pée, Karl-Heinz; Kalinowski, Jörn; Ludwig-Müller, Jutta; Wohlleben, Wolfgang

2017-09-10

Fungal aromatic polyketides display a very diverse and widespread group of natural products. Due to their excellent light absorption properties and widely studied biological activities, they offer numerous application for food, textile and pharmaceutical industry. The biosynthetic pathways of fungal aromatic polyketides usually involve a set of successive enzymes, in which a non-reductive polyketide synthase iteratively catalyzes the essential assembly of simple building blocks into (often polycyclic) aromatic compounds. However, only a limited number of such pathways have been described so far and further elucidation of the individual biosynthetic steps is needed to fully exploit the biotechnological and medicinal potential of these compounds. Here, we identified the bisanthraquinone skyrin as the main pigment of the fungus Cyanodermella asteris, an endophyte that has recently been isolated from the traditional Chinese medicinal plant Aster tataricus. The genome of C. asteris was sequenced, assembled and annotated, which enables first insights into a genome from a non-lichenized member of the class Lecanoromycetes. Genetic and in silico analyses led to the identification of a gene cluster of five genes suggested to encode the enzymatic pathway for skyrin. Our study is a starting point for rational pathway engineering in order to drive the production towards higher yields or more active derivatives. Moreover, our investigations revealed a large potential of secondary metabolite production in C. asteris as well as in all Lecanoromycetes of which genomes were available. These findings convincingly emphasize that Lecanoromycetes are prolific producers of secondary metabolites. Copyright © 2017 Elsevier B.V. All rights reserved.

A single active trehalose-6-P synthase (TPS) and a family of putative regulatory TPS-like proteins in Arabidopsis.

PubMed

Vandesteene, Lies; Ramon, Matthew; Le Roy, Katrien; Van Dijck, Patrick; Rolland, Filip

2010-03-01

Higher plants typically do not produce trehalose in large amounts, but their genome sequences reveal large families of putative trehalose metabolism enzymes. An important regulatory role in plant growth and development is also emerging for the metabolic intermediate trehalose-6-P (T6P). Here, we present an update on Arabidopsis trehalose metabolism and a resource for further detailed analyses. In addition, we provide evidence that Arabidopsis encodes a single trehalose-6-P synthase (TPS) next to a family of catalytically inactive TPS-like proteins that might fulfill specific regulatory functions in actively growing tissues.

Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

PubMed Central

Alquézar, Berta; Rodríguez, Ana; de la Peña, Marcos; Peña, Leandro

2017-01-01

Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z)-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays. PMID:28883829

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%.

United States Department of Agriculture-Agricultural Research Service research on natural products for pest management.

PubMed

Duke, Stephen O; Baerson, Scott R; Dayan, Franck E; Rimando, Agnes M; Scheffler, Brian E; Tellez, Mario R; Wedge, David E; Schrader, Kevin K; Akey, David H; Arthur, Frank H; De Lucca, Anthony J; Gibson, Donna M; Harrison, Howard F; Peterson, Joseph K; Gealy, David R; Tworkoski, Thomas; Wilson, Charles L; Morris, J Brad

2003-01-01

Recent research of the Agricultural Research Service of USDA on the use of natural products to manage pests is summarized. Studies of the use of both phytochemicals and diatomaceous earth to manage insect pests are discussed. Chemically characterized compounds, such as a saponin from pepper (Capsicum frutescens L), benzaldehyde, chitosan and 2-deoxy-D-glucose are being studied as natural fungicides. Resin glycosides for pathogen resistance in sweet potato and residues of semi-tropical leguminous plants for nematode control are also under investigation. Bioassay-guided isolation of compounds with potential use as herbicides or herbicide leads is underway at several locations. New natural phytotoxin molecular target sites (asparagine synthetase and fructose-1,6-bisphosphate aldolase) have been discovered. Weed control in sweet potato and rice by allelopathy is under investigation. Molecular approaches to enhance allelopathy in sorghum are also being undertaken. The genes for polyketide synthases involved in production of pesticidal polyketide compounds in fungi are found to provide clues for pesticide discovery. Gene expression profiles in response to fungicides and herbicides are being generated as tools to understand more fully the mode of action and to rapidly determine the molecular target site of new, natural fungicides and herbicides.

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.

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.

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.

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.

Improved docosahexaenoic acid production in Aurantiochytrium by glucose limited pH-auxostat fed-batch cultivation.

PubMed

Janthanomsuk, Panyawut; Verduyn, Cornelis; Chauvatcharin, Somchai

2015-11-01

Fed-batch, pH auxostat cultivation of the docosahexaenoic acid (DHA)-producing microorganism Aurantiochytrium B072 was performed to obtain high cell density and record high productivity of both total fatty acid (TFA) and DHA. Using glucose feeding by carbon excess (C-excess) and by C-limitation at various feeding rates (70%, 50% or 20% of C-excess), high biomass density was obtained and DHA/TFA content (w/w) was improved from 30% to 37% with a 50% glucose feed rate when compared with C-excess. To understand the biochemistry behind these improvements, lipogenic enzyme assays and in silico metabolic flux calculations were used and revealed that enzyme activity and C-fluxes to TFA were reduced with C-limited feeding but that the carbon flux to the polyketide synthase pathway increased relative to the fatty acid synthase pathway. As a result, a new strategy to improve the DHA to TFA content while maintaining relatively high DHA productivity is proposed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cyanobacteria: photosynthetic factories combining biodiversity, radiation resistance, and genetics to facilitate drug discovery.

PubMed

Cassier-Chauvat, Corinne; Dive, Vincent; Chauvat, Franck

2017-02-01

Cyanobacteria are ancient, abundant, and widely diverse photosynthetic prokaryotes, which are viewed as promising cell factories for the ecologically responsible production of chemicals. Natural cyanobacteria synthesize a vast array of biologically active (secondary) metabolites with great potential for human health, while a few genetic models can be engineered for the (low level) production of biofuels. Recently, genome sequencing and mining has revealed that natural cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The corresponding panoply of enzymes (polyketide synthases and non-ribosomal peptide synthases) of interest for synthetic biology can still be increased through gene manipulations with the tools available for the few genetically manipulable strains. In this review, we propose to exploit the metabolic diversity and radiation resistance of cyanobacteria, and when required the genetics of model strains, for the production and radioactive ( 14 C) labeling of bioactive products, in order to facilitate the screening for new drugs.

New Rimocidin/CE-108 Derivatives Obtained by a Crotonyl-CoA Carboxylase/Reductase Gene Disruption in Streptomyces diastaticus var. 108: Substrates for the Polyene Carboxamide Synthase PcsA

PubMed Central

Escudero, Leticia; Al-Refai, Mahmoud; Nieto, Cristina; Laatsch, Hartmut; Malpartida, Francisco; Seco, Elena M.

2015-01-01

The rimJ gene, which codes for a crotonyl-CoA carboxylase/reductase, lies within the biosynthetic gene cluster for two polyketides belonging to the polyene macrolide group (CE-108 and rimocidin) produced by Streptomyces diastaticus var. 108. Disruption of rimJ by insertional inactivation gave rise to a recombinant strain overproducing new polyene derivatives besides the parental CE-108 (2a) and rimocidin (4a). The structure elucidation of one of them, CE-108D (3a), confirmed the incorporation of an alternative extender unit for elongation step 13. Other compounds were also overproduced in the fermentation broth of rimJ disruptant. The new compounds are in vivo substrates for the previously described polyene carboxamide synthase PcsA. The rimJ disruptant strain, constitutively expressing the pcsA gene, allowed the overproduction of CE-108E (3b), the corresponding carboxamide derivative of CE-108D (3a), with improved pharmacological properties. PMID:26284936

Endoperoxide polyketides from a Chinese Plakortis simplex: further evidence of the impact of stereochemistry on antimalarial activity of simple 1,2-dioxanes.

PubMed

Chianese, Giuseppina; Persico, Marco; Yang, Fan; Lin, Hou-Wen; Guo, Yue-Wei; Basilico, Nicoletta; Parapini, Silvia; Taramelli, Donatella; Taglialatela-Scafati, Orazio; Fattorusso, Caterina

2014-09-01

Chemical investigation of the organic extract obtained from the sponge Plakortis simplex collected in the South China Sea afforded five new polyketide endoperoxides (2 and 4-7), along with two known analogues (1 and 3). The stereostructures of these metabolites have been deduced on the basis of spectroscopic analysis and chemical conversion. The isolated endoperoxide derivatives have been tested for their in vitro antimalarial activity against Plasmodium falciparum strains, showing IC50 values in the low micromolar range. The structure-activity relationships were analyzed by means of a detailed computational investigation and rationalized in the light of the mechanism of action proposed for this class of simple antimalarials. The relative orientation of the atoms involved in the putative radical generation and transfer reaction was demonstrated to have a great impact on the antimalarial activity. The resulting 3D pharmacophoric model can be a useful guide to design simple and effective antimalarial lead compounds belonging to the class of 1,2-dioxanes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Haplotype analysis of the germacrene A synthase gene and association with cynaropicrin content and biological activities in Cynara cardunculus.

PubMed

Ferro, Ana Margarida; Ramos, Patrícia; Guerra, Ângela; Parreira, Paula; Brás, Teresa; Guerreiro, Olinda; Jerónimo, Eliana; Capel, Carmen; Capel, Juan; Yuste-Lisbona, Fernando J; Duarte, Maria F; Lozano, Rafael; Oliveira, M Margarida; Gonçalves, Sónia

2018-04-01

Cynara cardunculus: L. represents a natural source of terpenic compounds, with the predominant molecule being cynaropicrin. Cynaropicrin is gaining interest since it has been correlated to anti-hyperlipidaemia, antispasmodic and cytotoxicity activity against leukocyte cancer cells. The objective of this work was to screen a collection of C. cardunculus, from different origins, for new allelic variants in germacrene A synthase (GAS) gene involved in the cynaropicrin biosynthesis and correlate them with improved cynaropicrin content and biological activities. Using high-resolution melting, nine haplotypes were identified. The putative impact of the identified allelic variants in GAS protein was evaluated by bioinformatic tools and polymorphisms that putatively lead to protein conformational changes were described. Additionally, cynaropicrin and main pentacyclic triterpenes contents, and antithrombin, antimicrobial and antiproliferative activities were also determined in C. cardunculus leaf lipophilic-derived extracts. In this work we identified allelic variants with putative impact on GAS protein, which are significantly associated with cynaropicrin content and antiproliferative activity. The results obtained suggest that the identified polymorphisms should be explored as putative genetic markers correlated with biological properties in Cynara cardunculus.

The Gα1-cAMP signaling pathway controls conidiation, development and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspora.

PubMed

Yu, Xi; Liu, Heng; Niu, Xueliang; Akhberdi, Oren; Wei, Dongsheng; Wang, Dan; Zhu, Xudong

2017-10-01

G-protein-mediated signaling pathways regulate fungal morphogenesis, development and secondary metabolism. In this study, we report a gene, pgα1, that putatively encodes the α-subunit of a group I G protein in Pestalotiopsis microspora NK17, which is known to produce various secondary metabolites, including the antitumor drug taxol and pestalotiollide B (PB). Mutants of pgα1 showed retarded vegetative growth, aging of the mycelium, premature conidiation, deformed conidia, significantly increased melanin production, and a sharp decrease in PB production. The introduction of extra copies of pgα1 led to a different phenotype that was characterized by enhanced production of PB. qRT-PCR revealed that the expression of pks1, which encodes melanin polyketide synthase, an enzyme that is involved in 1, 8-dihydroxynaphthalene (DHN) melanin biosynthesis, was up regulated by 55-fold in the absence of pgα1. Changes in conidiation and PB production in pgα1 mutants were able to be restored by the addition of exogenous cAMP. The deficiencies of PB production and conidiation in Δpgα1 were not able to be rescued by deletion or overexpression of a previously reported histone deacetylase gene (hid1), suggesting that pgα1 is able to override the effect of hid1 on PB production and conidiation. Our results suggested that the G protein-cAMP pathway plays a critical role in vegetative growth as well as in asexual development of P. microspora. Copyright © 2017 Elsevier GmbH. All rights reserved.

Identification and analysis of putative polyhydroxyalkanoate synthase (PhaC) in Pseudomonas fluorescens.

PubMed

Lim, Ju Hyoung; Rhie, Ho-Gun; Kim, Jeong Nam

2018-05-11

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. PHA granules mainly consisted of poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate- co -3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas . In vivo expression of the putative PHA synthase gene ( phaC Pf ) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli ( phaC Pf + , pUMS) grown in medium containing glucose accumulated PHA granules mainly consisting of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from E. coli fadR mutant ( phaC Pf + ) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaC Pf , suggesting that the putative PHA synthase of P. fluorescens mainly utilizes short-chain-length PHA precursors as a substrate.

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

Lessons from the synthetic chemist nature.

PubMed

Jürjens, Gerrit; Kirschning, Andreas; Candito, David A

2015-05-01

This conceptual review examines the ideal multistep synthesis from the perspective of nature. We suggest that besides step- and redox economies, one other key to efficiency is steady state processing with intermediates that are immediately transformed to the next intermediate when formed. We discuss four of nature's strategies (multicatalysis, domino reactions, iteration and compartmentation) that commonly proceed via short-lived intermediates and show that these strategies are also part of the chemist's portfolio. We particularly focus on compartmentation which in nature is found microscopically within cells (organelles) and between cells and on a molecular level on multiprotein scaffolds (e.g. in polyketide synthases) and demonstrate how compartmentation is manifested in modern multistep flow synthesis.

Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle

PubMed Central

Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G.; Köllner, Tobias G.

2016-01-01

Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

Chain elongation and cyclization in type III PKS DpgA.

PubMed

Wu, Hai-Chen; Li, Yi-San; Liu, Yu-Chen; Lyu, Syue-Yi; Wu, Chang-Jer; Li, Tsung-Lin

2012-04-16

Chain elongation and cyclization of precursors of dihydroxyphenylacetyl-CoA (DPA-CoA) catalyzed by the bacterial type III polyketide synthase DpgA were studied. Two labile intermediates, di- and tri-ketidyl-CoA (DK- and TK-CoA), were proposed and chemically synthesized. In the presence of DpgABD, each of these with [(13)C(3)]malonyl-CoA (MA-CoA) was able to form partially (13)C-enriched DPA-CoA. By NMR and MS analysis, the distribution of (13)C atoms in the partially (13)C-enriched DPA-CoA shed light on how the polyketide chain elongates and cyclizes in the DpgA-catalyzed reaction. Polyketone intermediates elongate in a manner different from that which had been believed: two molecules of DK-CoA, or one DK-CoA plus one acetoacetyl-CoA (AA-CoA), but not two molecules of AA-CoA can form one molecule of DPA-CoA. As a result, polyketidyl-CoA serves as both the starter and extender, whereas polyketone-CoA without the terminal carboxyl group can only act as an extender. The terminal carboxyl group is crucial for the cyclization that likely takes place on CoA. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

PubMed Central

Calderone, Christopher T.; Bumpus, Stefanie B.; Kelleher, Neil L.; Walsh, Christopher T.; Magarvey, Nathan A.

2008-01-01

The polyketide signaling metabolites bacillaene and dihydrobacillaene are biosynthesized in Bacillus subtilis on an enzymatic assembly line with both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules acting along with catalytic domains servicing the assembly line in trans. These signaling metabolites possess the unusual starter unit α-hydroxyisocaproate (α-HIC). We show here that it arises from initial activation of α-ketoisocaproate (α-KIC) by the first adenylation domain of PksJ (a hybrid PKS/NRPS) and installation on the pantetheinyl arm of the adjacent thiolation (T) domain. The α-KIC unit is elongated to α-KIC-Gly by the second NRPS module in PksJ as demonstrated by mass spectrometric analysis. The third module of PksJ uses PKS logic and contains an embedded ketoreductase (KR) domain along with two adjacent T domains. We show that this KR domain reduces canonical 3-ketobutyryl chains but also the α-keto group of α-KIC-containing intermediates on the PksJ T-domain doublet. This KR activity accounts for the α-HIC moiety found in the dihydrobacillaene/bacillaene pair and represents an example of an assembly-line dual-function α- and β-KR acting on disparate positions of a growing chain intermediate. PMID:18723688

Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif.

PubMed

Hurd, Matthew C; Kwon, Moonhyuk; Ro, Dae-Kyun

2017-08-26

Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX 8 W) motifs, and enzyme assays showed that the presence of both RRX 8 W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX 8 W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Ginger and turmeric expressed sequence tags identify signature genes for rhizome identity and development and the biosynthesis of curcuminoids, gingerols and terpenoids

PubMed Central

2013-01-01

Background Ginger (Zingiber officinale) and turmeric (Curcuma longa) accumulate important pharmacologically active metabolites at high levels in their rhizomes. Despite their importance, relatively little is known regarding gene expression in the rhizomes of ginger and turmeric. Results In order to identify rhizome-enriched genes and genes encoding specialized metabolism enzymes and pathway regulators, we evaluated an assembled collection of expressed sequence tags (ESTs) from eight different ginger and turmeric tissues. Comparisons to publicly available sorghum rhizome ESTs revealed a total of 777 gene transcripts expressed in ginger/turmeric and sorghum rhizomes but apparently absent from other tissues. The list of rhizome-specific transcripts was enriched for genes associated with regulation of tissue growth, development, and transcription. In particular, transcripts for ethylene response factors and AUX/IAA proteins appeared to accumulate in patterns mirroring results from previous studies regarding rhizome growth responses to exogenous applications of auxin and ethylene. Thus, these genes may play important roles in defining rhizome growth and development. Additional associations were made for ginger and turmeric rhizome-enriched MADS box transcription factors, their putative rhizome-enriched homologs in sorghum, and rhizomatous QTLs in rice. Additionally, analysis of both primary and specialized metabolism genes indicates that ginger and turmeric rhizomes are primarily devoted to the utilization of leaf supplied sucrose for the production and/or storage of specialized metabolites associated with the phenylpropanoid pathway and putative type III polyketide synthase gene products. This finding reinforces earlier hypotheses predicting roles of this enzyme class in the production of curcuminoids and gingerols. Conclusion A significant set of genes were found to be exclusively or preferentially expressed in the rhizome of ginger and turmeric. Specific transcription factors and other regulatory genes were found that were common to the two species and that are excellent candidates for involvement in rhizome growth, differentiation and development. Large classes of enzymes involved in specialized metabolism were also found to have apparent tissue-specific expression, suggesting that gene expression itself may play an important role in regulating metabolite production in these plants. PMID:23410187

Biosynthesis of t-Butyl in Apratoxin A: Functional Analysis and Architecture of a PKS Loading Module.

PubMed

Skiba, Meredith A; Sikkema, Andrew P; Moss, Nathan A; Lowell, Andrew N; Su, Min; Sturgis, Rebecca M; Gerwick, Lena; Gerwick, William H; Sherman, David H; Smith, Janet L

2018-05-08

The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.

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.

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.

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.

Microbial Flora Associated with the Halophyte–Salsola imbricate and Its Biotechnical Potential

PubMed Central

Bibi, Fehmida; Strobel, Gary A.; Naseer, Muhammad I.; Yasir, Muhammad; Khalaf Al-Ghamdi, Ahmed A.; Azhar, Esam I.

2018-01-01

Halophytes are associated with the intertidal forest ecosystem of Saudi Arabia and seemingly have an immense potential for yielding useful and important natural products. In this study we have aimed to isolate and characterize the endophytic and rhizospheric bacterial communities from the halophyte, Salsola imbricata, In addition these bacterial strains were identified and selected strains were further studied for bioactive secondary metabolites. At least 168 rhizspheric and endophytic bacteria were isolated and of these 22 were active antagonists against the oomycetous fungal plant pathogens, Phytophthora capsici and Pythium ultimum. Active cultures were mainly identified with molecular techniques (16S r DNA) and this revealed 95.7–100% sequence similarities with relevant type strains. These microorgansims were grouped into four major classes: Actinobacteria, Firmicutes, β-Proteobacteria, and γ-Proteobacteria. Production of fungal cell wall lytic enzymes was detected mostly in members of Actinobacteria and Firmicutes. PCR screening for type I polyketide synthases (PKS-I), type II polyketide synthases (PKS-II) and nonribosomal peptide synthetases (NRPS) revealed 13 of the 22 strains (59%) were positive for at least one of these important biosynthetic genes that are known to be involved in the synthesis of important antibiotics. Four bacterial strains of Actinobacteria with potential antagonistic activity including two rhizobacteria, EA52 (Nocardiopsis sp.), EA58 (Pseudonocardia sp.) and two endophytic bacteria Streptomyces sp. (EA65) and Streptomyces sp. (EA67) were selected for secondary metabolite analyses using LC-MS. As a result, the presence of different bioactive compounds in the culture extracts was detected some of which are already reported for their diverse biological activities including antibiotics such as Sulfamethoxypyridazine, Sulfamerazine, and Dimetridazole. In conclusion, this study provides an insight into antagonistic bacterial population especially the Actinobacteria from S. imbricata, producing antifungal metabolites of medical significance and characterized taxonomically in future. PMID:29445362

Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis.

PubMed

Sitthithaworn, W; Kojima, N; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Noji, M; Saito, K; Niwa, Y; Sankawa, U

2001-02-01

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method. Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence. When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP). The structural factors determining the product length in plant GGPPSs were investigated by constructing S. dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase. The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length. Further, when a chimeric gene comprised of the putative transit peptide of the S. dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S. dulcis GGPPS is a chloroplast protein.

Phytoalexins of the Pyrinae: Biphenyls and dibenzofurans

PubMed Central

Chizzali, Cornelia

2012-01-01

Summary Biphenyls and dibenzofurans are the phytoalexins of the Pyrinae, a subtribe of the plant family Rosaceae. The Pyrinae correspond to the long-recognized Maloideae. Economically valuable species of the Pyrinae are apples and pears. Biphenyls and dibenzofurans are formed de novo in response to infection by bacterial and fungal pathogens. The inducible defense compounds were also produced in cell suspension cultures after treatment with biotic and abiotic elicitors. The antimicrobial activity of the phytoalexins was demonstrated. To date, 10 biphenyls and 17 dibenzofurans were isolated from 14 of the 30 Pyrinae genera. The most widely distributed compounds are the biphenyl aucuparin and the dibenzofuran γ-cotonefuran. The biosynthesis of the two classes of defense compounds is not well understood, despite the importance of the fruit crops. More recent studies have revealed simultaneous accumulation of biphenyls and dibenzofurans, suggesting sequential, rather than the previously proposed parallel, biosynthetic pathways. Elicitor-treated cell cultures of Sorbus aucuparia served as a model system for studying phytoalexin metabolism. The key enzyme that forms the carbon skeleton is biphenyl synthase. The starter substrate for this type-III polyketide synthase is benzoyl-CoA. In apples, biphenyl synthase is encoded by a gene family, members of which are differentially regulated. Metabolism of the phytoalexins may provide new tools for designing disease control strategies for fruit trees of the Pyrinae subtribe. PMID:22563359

Amino Acid Precursor Supply in the Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin by Streptomyces lydicus▿†

PubMed Central

Gómez, Cristina; Horna, Dina H.; Olano, Carlos; Palomino-Schätzlein, Martina; Pineda-Lucena, Antonio; Carbajo, Rodrigo J.; Braña, Alfredo F.; Méndez, Carmen; Salas, José A.

2011-01-01

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin. PMID:21665968

Amino acid precursor supply in the biosynthesis of the RNA polymerase inhibitor streptolydigin by Streptomyces lydicus.

PubMed

Gómez, Cristina; Horna, Dina H; Olano, Carlos; Palomino-Schätzlein, Martina; Pineda-Lucena, Antonio; Carbajo, Rodrigo J; Braña, Alfredo F; Méndez, Carmen; Salas, José A

2011-08-01

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin.

Multiplex Detection of Aspergillus Species.

PubMed

Martínez-Culebras, Pedro; Selma, María Victoria; Aznar, Rosa

2017-01-01

Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied in wine grapes. It includes a pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors.

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.

Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.

PubMed

Jin, Zhehao; Kim, Jin-Hee; Park, Sang Un; Kim, Soo-Un

2016-12-01

Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

Removal of a putative inhibitory element reduces the calcium-dependent calmodulin activation of neuronal nitric-oxide synthase.

PubMed

Montgomery, H J; Romanov, V; Guillemette, J G

2000-02-18

Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.

The Putative Protein Methyltransferase LAE1 of Trichoderma atroviride Is a Key Regulator of Asexual Development and Mycoparasitism

PubMed Central

Aghcheh, Razieh Karimi; Druzhinina, Irina S.; Kubicek, Christian P.

2013-01-01

In Ascomycota the protein methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus Trichoderma atroviride is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order to learn the possible involvement of LAE1 in these two traits, we assessed the effect of deletion and overexpression of lae1 gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a Δ lae1 and 30–50% increased in lae1-overexpressing (OElae1) strains. In darkness, Δ lae1 strains did not sporulate, and the OElae1 strains produced as much spores as the parent strain. Loss-of-function of lae1 also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of lae1 also increased the sensitivity of T. atroviride to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the OElae1 strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the Δ lae1 strain correlated with a significant underexpressionn of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 ß-glucanases, polyketide synthases and small cystein-rich secreted proteins), which in turn was reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Our study shows T. atroviride LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi. PMID:23826217

Anti-MRSA and anti-TB metabolites from marine-derived Verrucosispora sp. MS100047.

PubMed

Huang, Pei; Xie, Feng; Ren, Biao; Wang, Qian; Wang, Jian; Wang, Qi; Abdel-Mageed, Wael M; Liu, Miaomiao; Han, Jianying; Oyeleye, Ayokunmi; Shen, Jinzhao; Song, Fuhang; Dai, Huanqin; Liu, Xueting; Zhang, Lixin

2016-09-01

Microbes belonging to the genus Verrucosispora possess significant chemical diversity and biological properties. They have attracted the interests of many researchers and are becoming promising resources in the marine natural product research field. A bioassay-guided isolation from the crude extract of Verrucosispora sp. strain MS100047, isolated from sediments collected from the South China Sea, has led to the identification of a new salicylic derivative, glycerol 1-hydroxy-2,5-dimethyl benzoate (1), along with three known compounds, brevianamide F (2), abyssomicin B (3), and proximicin B (4). Compound 1 showed selective activity against methicillin-resistant Staphylococcus aureus (MRSA) with a minimum inhibitory concentration (MIC) value of 12.5 μg/mL. Brevianamide F (2), which was isolated from actinomycete for the first time, showed a good anti-BCG activity with a MIC value of 12.5 μg/mL that has not been reported previously in literatures. Proximicin B (4) showed significant anti-MRSA (MIC = 3.125 μg/mL), anti-BCG (MIC = 6.25 μg/mL), and anti-tuberculosis (TB) (MIC = 25 μg/mL) activities. This is the first report on the anti-tubercular activities of proximicins. In addition, Verrucosispora sp. strain MS100047 was found to harbor 18 putative secondary metabolite gene clusters based on genomic sequence analysis. These include the biosynthetic loci encoding polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) consistent with abyssomicins and proximicins, respectively. The biosynthetic pathways of these isolated compounds have been proposed. These results indicate that MS100047 possesses a great potential as a source of active secondary metabolites.

Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin.

PubMed

Zhang, Ji; An, Jing; Wang, Ji-Jia; Yan, Yi-Jun; He, Hai-Rong; Wang, Xiang-Jing; Xiang, Wen-Sheng

2013-12-01

Milbemycins A3/A4 are important 16-membered macrolides which have been commercialized and widely used as pesticide and veterinary medicine. However, similar to other milbemycin producers, the production of milbemycins A3/A4 in Streptomyces bingchenggensis is usually accompanied with undesired by-products such as C5-O - methylmilbemycins B2/B3 (α-class) and β1/β2 (β-class) together with nanchangmycin. In order to obtain high yield milbemycins A3/A4-producing strains that produce milbemycins A3/A4 as main components, milD, a putative C5-O-methyltransferase gene of S. bingchenggensis , was biofunctionally investigated by heterologous expression in Escherichia coli . Enzymatic analysis indicated that MilD can catalyze both α-class (A3/A4) and β-class milbemycins (β11) into C5-O-methylmilbemycins B2/B3 and β1, respectively, suggesting little effect of furan ring formed between C6 and C8a on the C5-O-methylation catalyzed by MilD. Deletion of milD gene resulted in the elimination of C5-Omethylmilbemycins B2/B3 and β1/β2 together with an increased yield of milbemycins A3/A4 in disruption strain BCJ13. Further disruption of the gene nanLD encoding loading module of polyketide synthase responsible for the biosynthesis of nanchangmycin led to strain BCJ36 that abolished the production of nanchangmycin. Importantly, mutant strain BCJ36 (ΔmilDΔnanLD) produced milbemycins A3/A4 as main secondary metabolites with a yield of 2312 ± 47 μg/ml, which was approximately 74 % higher than that of the initial strain S. bingchenggensis BC-109-6 (1326 ± 37 μg/ml).

The Biogeography of Putative Microbial Antibiotic Production

PubMed Central

Bryant, Jessica A.; Charkoudian, Louise K.; Docherty, Kathryn M.; Jones, Evan; Kembel, Steven W.; Green, Jessica L.; Bohannan, Brendan J. M.

2015-01-01

Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases.

PubMed

Parveen, Iffat; Wang, Mei; Zhao, Jianping; Chittiboyina, Amar G; Tabanca, Nurhayat; Ali, Abbas; Baerson, Scott R; Techen, Natascha; Chappell, Joe; Khan, Ikhlas A; Pan, Zhiqiang

2015-11-01

Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

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.

Tomato Cutin Deficient 1 (CD1) and Putative Orthologs Comprise an Ancient Family of Cutin Synthase-like (CUS) Proteins that are Conserved among Land Plants

PubMed Central

Yeats, Trevor H.; Huang, Wenlin; Chatterjee, Subhasish; Viart, Hélène M-F.; Clausen, Mads H.; Stark, Ruth E.; Rose, Jocelyn K.C.

2014-01-01

Summary The aerial epidermis of all land plants is covered with a hydrophobic cuticle that provides essential protection from desiccation, and so its evolution is believed to have been prerequisite for terrestrial colonization. A major structural component of apparently all plant cuticles is cutin, a polyester of hydroxy fatty acids. However, despite its ubiquity, the details of cutin polymeric structure and the mechanisms of its formation and remodeling are not well understood. We recently reported that cutin polymerization in tomato (Solanum lycopersicum) fruit occurs via transesterification of hydroxyacylglycerol precursors, catalyzed by the GDSL-motif lipase/hydrolase family protein (GDSL) Cutin Deficient 1 (CD1). Here we present additional biochemical characterization of CD1 and putative orthologs from Arabidopsis thaliana and the moss Physcomitrella patens, which represent a distinct clade of cutin synthases within the large GDSL super-family. We demonstrate that members of this ancient and conserved family of cutin synthase-like (CUS) proteins act as polyester synthases with negligible hydrolytic activity. Moreover, solution-state NMR analysis indicates that CD1 catalyzes the formation of primarily linear cutin oligomeric products in vitro. These results reveal a conserved mechanism of cutin polyester synthesis in land plants, and suggest that elaborations of the linear polymer, such as branching or cross-linking, may require additional, as yet unknown, factors. PMID:24372802

The First Prokaryotic Trehalose Synthase Complex Identified in the Hyperthermophilic Crenarchaeon Thermoproteus tenax

PubMed Central

Bräsen, Christopher; Hensel, Reinhard; Lupas, Andrei N.; Brinkmann, Henner; Siebers, Bettina

2013-01-01

The role of the disaccharide trehalose, its biosynthesis pathways and their regulation in Archaea are still ambiguous. In Thermoproteus tenax a fused trehalose-6-phosphate synthase/phosphatase (TPSP), consisting of an N-terminal trehalose-6-phosphate synthase (TPS) and a C-terminal trehalose-6-phosphate phosphatase (TPP) domain, was identified. The tpsp gene is organized in an operon with a putative glycosyltransferase (GT) and a putative mechanosensitive channel (MSC). The T. tenax TPSP exhibits high phosphatase activity, but requires activation by the co-expressed GT for bifunctional synthase-phosphatase activity. The GT mediated activation of TPS activity relies on the fusion of both, TPS and TPP domain, in the TPSP enzyme. Activation is mediated by complex-formation in vivo as indicated by yeast two-hybrid and crude extract analysis. In combination with first evidence for MSC activity the results suggest a sophisticated stress response involving TPSP, GT and MSC in T. tenax and probably in other Thermoproteales species. The monophyletic prokaryotic TPSP proteins likely originated via a single fusion event in the Bacteroidetes with subsequent horizontal gene transfers to other Bacteria and Archaea. Furthermore, evidence for the origin of eukaryotic TPSP fusions via HGT from prokaryotes and therefore a monophyletic origin of eukaryotic and prokaryotic fused TPSPs is presented. This is the first report of a prokaryotic, archaeal trehalose synthase complex exhibiting a much more simple composition than the eukaryotic complex described in yeast. Thus, complex formation and a complex-associated regulatory potential might represent a more general feature of trehalose synthesizing proteins. PMID:23626675

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.

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha[OPEN

PubMed Central

Zhuang, Xun; Jiang, Zuodong; Jia, Qidong; Babbitt, Patricia C.

2016-01-01

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases. PMID:27650333

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

Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower (Helianthus annuus L., Asteraceae).

PubMed

Aschenbrenner, Anna-Katharina; Kwon, Moonhyuk; Conrad, Jürgen; Ro, Dae-Kyun; Spring, Otmar

2016-04-01

Sunflower is known to produce a variety of bisabolene-type sesquiterpenes and accumulates these substances in trichomes of leaves, stems and flowering parts. A bioinformatics approach was used to identify the enzyme responsible for the initial step in the biosynthesis of these compounds from its precursor farnesyl pyrophosphate. Based on sequence similarity with a known bisabolene synthases from Arabidopsis thaliana AtTPS12, candidate genes of Helianthus were searched in EST-database and used to design specific primers. PCR experiments identified two candidates in the RNA pool of linear glandular trichomes of sunflower. Their sequences contained the typical motifs of sesquiterpene synthases and their expression in yeast functionally characterized them as bisabolene synthases. Spectroscopic analysis identified the stereochemistry of the product of both enzymes as (Z)-γ-bisabolene. The origin of the two sunflower bisabolene synthase genes from the transcripts of linear trichomes indicates that they may be involved in the synthesis of sesquiterpenes produced in these trichomes. Comparison of the amino acid sequences of the sunflower bisabolene synthases showed high similarity with sesquiterpene synthases from other Asteracean species and indicated putative evolutionary origin from a β-farnesene synthase. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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.

Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.).

PubMed

Yamaguchi, S; Saito, T; Abe, H; Yamane, H; Murofushi, N; Kamiya, Y

1996-08-01

The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region.

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

PubMed

Yao, Lin; Tan, Chong; Song, Jinzhu; Yang, Qian; Yu, Lijie; Li, Xinling

2016-01-01

Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669bp) and pksT-2 (7901bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase-acyltransferase domains through Agrobacterium-mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

PubMed

Klein, B; Pawlowski, K; Höricke-Grandpierre, C; Schell, J; Töpfer, R

1992-05-01

A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata. This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity. Hybridization studies revealed that in C. lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

Potent Nematicidal Activity and New Hybrid Metabolite Production by Disruption of a Cytochrome P450 Gene Involved in the Biosynthesis of Morphological Regulatory Arthrosporols in Nematode-Trapping Fungus Arthrobotrys oligospora.

PubMed

Song, Tian-Yang; Xu, Zi-Fei; Chen, Yong-Hong; Ding, Qiu-Yan; Sun, Yu-Rong; Miao, Yang; Zhang, Ke-Qin; Niu, Xue-Mei

2017-05-24

Types of polyketide synthase-terpenoid synthase (PKS-TPS) hybrid metabolites, including arthrosporols with significant morphological regulatory activity, have been elucidated from nematode-trapping fungus Arthrobotrys oligospora. A previous study suggested that the gene cluster AOL_s00215 in A. oligospora was involved in the production of arthrosporols. Here, we report that disruption of one cytochrome P450 monooxygenase gene AOL_s00215g280 in the cluster resulted in significant phenotypic difference and much aerial hyphae. A further bioassay indicated that the mutant showed a dramatic decrease in the conidial formation but developed numerous traps and killed 85% nematodes within 6 h in contact with prey, in sharp contrast to the wild-type strain with no obvious response. Chemical investigation revealed huge accumulation of three new PKS-TPS epoxycyclohexone derivatives with different oxygenated patterns around the epoxycyclohexone moiety and the absence of arthrosporols in the cultural broth of the mutant ΔAOL_s00215g280. These findings suggested that a study on the biosynthetic pathway for morphological regulatory metabolites in nematode-trapping fungus would provide an efficient way to develop new fungal biocontrol agents.

Computer Simulation of the Virulome of Bacillus anthracis Using Proteomics

DTIC Science & Technology

2006-07-31

hypothetical protein gi|47526566 spermidine /putrescine ABC transporter, spermidine /putrescine-binding protein gi|47526625 oligoendopeptidase F, putative gi...glutamyl-trna(gln) amidotransferase, a subunit x gi|50196927 aspartate aminotransferase x gi|50196970 spermidine synthase x

Fungi on the Skin: Dermatophytes and Malassezia

PubMed Central

White, Theodore C.; Findley, Keisha; Dawson, Thomas L.; Scheynius, Annika; Boekhout, Teun; Cuomo, Christina A.; Xu, Jun; Saunders, Charles W.

2014-01-01

Several human skin diseases and disorders are associated with two groups of fungi, the dermatophytes and Malassezia. Although these skin-related problems are not generally life threatening, they are among the most common diseases and disorders of mankind. These fungi are phylogenetically divergent, with the dermatophytes within the Ascomycota and Malassezia within Basidiomycota. Genome analysis indicates that the adaptations to the skin environment are different in these two groups of fungi. Malassezia are dependent on host lipids and secrete lipases and phospholipases that likely release host fatty acids. The dermatophytes encode multiple enzymes with potential roles in modulating host interactions: polyketide synthases, nonribosomal peptide synthetases, LysM, proteases, kinases, and pseudokinases. These two fungal groups have maximized their interactions with the host using two very different mechanisms. PMID:25085959

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis.

PubMed

Toporkova, Yana Y; Gorina, Svetlana S; Bessolitsyna, Elena K; Smirnova, Elena O; Fatykhova, Valeria S; Brühlmann, Fredi; Ilyina, Tatiana M; Mukhtarova, Lucia S; Grechkin, Alexander N

2018-04-01

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the "hydroperoxide-binding domain" (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs. Copyright © 2018 Elsevier B.V. All rights reserved.

Origin of the Allyl Group in FK506 Biosynthesis*

PubMed Central

Goranovič, Dušan; Kosec, Gregor; Mrak, Peter; Fujs, Štefan; Horvat, Jaka; Kuščer, Enej; Kopitar, Gregor; Petković, Hrvoje

2010-01-01

FK506 (tacrolimus) is a secondary metabolite with a potent immunosuppressive activity, currently registered for use as immunosuppressant after organ transplantation. FK506 and FK520 are biogenetically related natural products that are synthesized by combined polyketide synthase/nonribosomal peptide synthetase systems. The entire gene cluster for biosynthesis of FK520 from Streptomyces hygroscopicus var. ascomyceticus has been cloned and sequenced. On the other hand, the FK506 gene cluster from Streptomyces sp. MA6548 (ATCC55098) was sequenced only partially, and it was reasonable to expect that additional genes would be required for the provision of substrate supply. Here we report the identification of a previously unknown region of the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 containing genes encoding the provision of unusual building blocks for FK506 biosynthesis as well as a regulatory gene. Among others, we identified a group of genes encoding biosynthesis of the extender unit that forms the allyl group at carbon 21 of FK506. Interestingly, we have identified a small independent diketide synthase system involved in the biosynthesis of the allyl group. Inactivation of one of these genes, encoding an unusual ketosynthase domain, resulted in an FK506 nonproducing strain, and the production was restored when a synthetic analog of the allylmalonyl-CoA extender unit was added to the cultivation medium. Based on our results, we propose a biosynthetic pathway for the provision of an unusual five-carbon extender unit, which is carried out by a novel diketide synthase complex. PMID:20194504

Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis

PubMed Central

de Lima, Júlio C.; de Costa, Fernanda; Füller, Thanise N.; Rodrigues-Corrêa, Kelly C. da Silva; Kerber, Magnus R.; Lima, Mariano S.; Fett, Janette P.; Fett-Neto, Arthur G.

2016-01-01

Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes) and rosin (diterpenes) fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta β-pinene synthase [PcTPS-(−)β-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1), Pinus taeda α-pinene synthase [PtTPS-(+)αpin], and P. taeda α-farnesene synthase (PtαFS) were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers. PMID:27379135

Reference Genes for qPCR Analysis in Resin-Tapped Adult Slash Pine As a Tool to Address the Molecular Basis of Commercial Resinosis.

PubMed

de Lima, Júlio C; de Costa, Fernanda; Füller, Thanise N; Rodrigues-Corrêa, Kelly C da Silva; Kerber, Magnus R; Lima, Mariano S; Fett, Janette P; Fett-Neto, Arthur G

2016-01-01

Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes) and rosin (diterpenes) fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta β-pinene synthase [PcTPS-(-)β-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1), Pinus taeda α-pinene synthase [PtTPS-(+)αpin], and P. taeda α-farnesene synthase (PtαFS) were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers.

A Chitin Synthase and Its Regulator Protein Are Critical for Chitosan Production and Growth of the Fungal Pathogen Cryptococcus neoformans†

PubMed Central

Banks, Isaac R.; Specht, Charles A.; Donlin, Maureen J.; Gerik, Kimberly J.; Levitz, Stuart M.; Lodge, Jennifer K.

2005-01-01

Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30°C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37°C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Δ and the csr2Δ mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target. PMID:16278457

A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans.

PubMed

Banks, Isaac R; Specht, Charles A; Donlin, Maureen J; Gerik, Kimberly J; Levitz, Stuart M; Lodge, Jennifer K

2005-11-01

Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30 degrees C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37 degrees C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Delta and the csr2Delta mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target.

Tomato Cutin Deficient 1 (CD1) and putative orthologs comprise an ancient family of cutin synthase-like (CUS) proteins that are conserved among land plants.

PubMed

Yeats, Trevor H; Huang, Wenlin; Chatterjee, Subhasish; Viart, Hélène M-F; Clausen, Mads H; Stark, Ruth E; Rose, Jocelyn K C

2014-03-01

The aerial epidermis of all land plants is covered with a hydrophobic cuticle that provides essential protection from desiccation, and so its evolution is believed to have been prerequisite for terrestrial colonization. A major structural component of apparently all plant cuticles is cutin, a polyester of hydroxy fatty acids; however, despite its ubiquity, the details of cutin polymeric structure and the mechanisms of its formation and remodeling are not well understood. We recently reported that cutin polymerization in tomato (Solanum lycopersicum) fruit occurs via transesterification of hydroxyacylglycerol precursors, catalyzed by the GDSL-motif lipase/hydrolase family protein (GDSL) Cutin Deficient 1 (CD1). Here, we present additional biochemical characterization of CD1 and putative orthologs from Arabidopsis thaliana and the moss Physcomitrella patens, which represent a distinct clade of cutin synthases within the large GDSL superfamily. We demonstrate that members of this ancient and conserved family of cutin synthase-like (CUS) proteins act as polyester synthases with negligible hydrolytic activity. Moreover, solution-state NMR analysis indicates that CD1 catalyzes the formation of primarily linear cutin oligomeric products in vitro. These results reveal a conserved mechanism of cutin polyester synthesis in land plants, and suggest that elaborations of the linear polymer, such as branching or cross-linking, may require additional, as yet unknown, factors. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

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.

Evaluation of the Antimicrobial Activity of Endophytic Bacterial Populations From Chinese Traditional Medicinal Plant Licorice and Characterization of the Bioactive Secondary Metabolites Produced by Bacillus atrophaeus Against Verticillium dahliae.

PubMed

Mohamad, Osama A A; Li, Li; Ma, Jin-Biao; Hatab, Shaimaa; Xu, Lin; Guo, Jian-Wei; Rasulov, Bakhtiyor A; Liu, Yong-Hong; Hedlund, Brian P; Li, Wen-Jun

2018-01-01

Endophytic bacteria associated with medicinal plants possess unique strategies that enhance growth and suvival of host plants, many of which are mediated by distinctive secondary metabolites. These bacteria and their secondary metabolites are important subjects for both basic and applied research aimed at sustainable agriculture. In the present study, 114 endophytic strains isolated from the wild ethnomedicinal plant Glycyrrhiza uralensis (licorice) were screened for their in vitro antimicrobial activities against common fungal pathogens of tomato ( Fusarium oxysporum f. sp., Fulvia fulva , Alternaria solani ), cotton ( Fusarium oxysporum f. sp. Vesinfectum, Verticillium dahliae ), pomegranite ( Ceratocystis fimbriata ), Cymbidinium ( Colletotrichum gloeosporioides ), and Tsao-ko ( Pestalotiopsis microspora and Fusarium graminearum ) and the common bacteria Staphylococcus aureus , Bacillus cereus , Salmonella enteritidis , and Escherichia coli . Several Bacillus strains, particularly Bacillus atrophaeus and Bacillus mojavensis , had a broad spectrum of antifungal and antibacterial activity. A total of 16 strains, selected based on broad antimicrobial activity, were shown to contain at least one putative secondary metabolite-encoding gene (i.e., polyketide synthase or non-ribosomal peptide synthetase) and/or one lytic enzyme (i.e., protease, cellulase, lipase, chitinase), which may be important mediators of antagonistic activity against pathogens. Five strains, representing Bacillus atrophaeus and Bacillus mojavensis , were selected for plant growth chamber experiments based on strong in vitro antifungal activities. All five strains significantly reduced disease severity in Arabidopsis thaliana plants challenged with V. dahlia infection. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of Bacillus atrophaeus strain XEGI50 showed that at least 13 compounds were produced only during co-cultivation with V. dahlia , including putative compounds known to have antimicrobial activity, such as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; 9,12-octadecadienoic acid (Z,Z)-, methyl ester; 9-octadecenoic acid, methyl ester, (E)-; and decanedioic acid, bis(2-ethylhexyl) ester. To our knowledge, this study is the first to report that bacteria isolated from G. uralensis have biocontrol abilities. Our findings provide new insights into the antimicrobial activities of natural endophytes, particularly B. atrophaeus , and suggest this species may a promising candidate as a biocontrol agent to confer resistance to Verticillium wilt disease and other phytopathogens in cotton and other crops.

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.

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.

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.

Fungi on the skin: dermatophytes and Malassezia.

PubMed

White, Theodore C; Findley, Keisha; Dawson, Thomas L; Scheynius, Annika; Boekhout, Teun; Cuomo, Christina A; Xu, Jun; Saunders, Charles W

2014-08-01

Several human skin diseases and disorders are associated with two groups of fungi, the dermatophytes and Malassezia. Although these skin-related problems are not generally life threatening, they are among the most common diseases and disorders of mankind. These fungi are phylogenetically divergent, with the dermatophytes within the Ascomycota and Malassezia within Basidiomycota. Genome analysis indicates that the adaptations to the skin environment are different in these two groups of fungi. Malassezia are dependent on host lipids and secrete lipases and phospholipases that likely release host fatty acids. The dermatophytes encode multiple enzymes with potential roles in modulating host interactions: polyketide synthases, nonribosomal peptide synthetases, LysM, proteases, kinases, and pseudokinases. These two fungal groups have maximized their interactions with the host using two very different mechanisms. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

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.

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.

Volatile emissions of scented Alstroemeria genotypes are dominated by terpenes, and a myrcene synthase gene is highly expressed in scented Alstroemeria flowers

PubMed Central

Aros, Danilo; Gonzalez, Veronica; Allemann, Rudolf K.; Müller, Carsten T.; Rosati, Carlo; Rogers, Hilary J.

2012-01-01

Native to South America, Alstroemeria flowers are known for their colourful tepals, and Alstroemeria hybrids are an important cut flower. However, in common with many commercial cut flowers, virtually all the commercial Alstroemeria hybrids are not scented. The cultivar ‘Sweet Laura’ is one of very few scented commercial Alstroemeria hybrids. Characterization of the volatile emission profile of these cut flowers revealed three major terpene compounds: (E)-caryophyllene, humulene (also known as α-caryophyllene), an ocimene-like compound, and several minor peaks, one of which was identified as myrcene. The profile is completely different from that of the parental scented species A. caryophyllaea. Volatile emission peaked at anthesis in both scented genotypes, coincident in cv. ‘Sweet Laura’ with the maximal expression of a putative terpene synthase gene AlstroTPS. This gene was preferentially expressed in floral tissues of both cv. ‘Sweet Laura’ and A. caryophyllaea. Characterization of the AlstroTPS gene structure from cv. ‘Sweet Laura’ placed it as a member of the class III terpene synthases, and the predicted 567 amino acid sequence placed it into the subfamily TPS-b. The conserved sequences R28(R)X8W and D321DXXD are the putative Mg2+-binding sites, and in vitro assay of AlstroTPS expressed in Escherichia coli revealed that the encoded enzyme possesses myrcene synthase activity, consistent with a role for AlstroTPS in scent production in Alstroemeria cv. ‘Sweet Laura’ flowers. PMID:22268153

Identification and characterization of a class III chitin synthase gene of Moniliophthora perniciosa, the fungus that causes witches' broom disease of cacao.

PubMed

Souza, Catiane S; Oliveira, Bruno M; Costa, Gustavo G L; Schriefer, Albert; Selbach-Schnadelbach, Alessandra; Uetanabaro, Ana Paula T; Pirovani, Carlos P; Pereira, Gonçalo A G; Taranto, Alex G; Cascardo, Júlio Cézar de M; Góes-Neto, Aristóteles

2009-08-01

Chitin synthase (CHS) is a glucosyltransferase that converts UDP-N-acetylglucosamine into chitin, one of the main components of fungal cell wall. Class III chitin synthases act directly in the formation of the cell wall. They catalyze the conversion of the immediate precursor of chitin and are responsible for the majority of chitin synthesis in fungi. As such, they are highly specific molecular targets for drugs that can inhibit the growth and development of fungal pathogens. In this work, we have identified and characterized a chitin synthase gene of Moniliophthora perniciosa (Mopchs) by primer walking. The complete gene sequence is 3,443 bp, interrupted by 13 small introns, and comprises a cDNA with an ORF with 2,739 bp, whose terminal region was experimentally determined, encoding a protein with 913 aa that harbors all the motifs and domains typically found in class III chitin synthases. This is the first report on the characterization of a chitin synthase gene, its mature transcription product, and its putative protein in basidioma and secondary mycelium stages of M. perniciosa, a basidiomycotan fungus that causes witches' broom disease of cacao.

Transcriptome Analysis Revealed Highly Expressed Genes Encoding Secondary Metabolite Pathways and Small Cysteine-Rich Proteins in the Sclerotium of Lignosus rhinocerotis

PubMed Central

Yap, Hui-Yeng Y.; Chooi, Yit-Heng; Fung, Shin-Yee; Ng, Szu-Ting; Tan, Chon-Seng; Tan, Nget-Hong

2015-01-01

Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications. PMID:26606395

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

Guo, Chun-Jun; Sun, Wei-Wen; Bruno, Kenneth S.

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes or non-ribosomal peptide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. More interestingly, further experiments revealedmore » that the aspulvinone E produced by two different genes accumulates in different fungal compartments. And this spatial control of aspulvinone E production is likely to be regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is inserted in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. The study also identified one trans-prenyltransferase AbpB which is capable of prenylating two different substrates aspulvinones and butyrolactones. In total, our study shows the first example in which the locally distribution of the same natural product could lead to its incorporation into different SM pathways.« less

[Isolation of actinobacteria with antibiotic associated with soft coral Nephthea sp].

PubMed

Ma, Liang; Zhang, Wenjun; Zhu, Yiguang; Wu, Zhengchao; Saurav, Kumar; Hang, Hui; Zhang, Changsheng

2013-10-04

The present study aims to isolate and identify actinobacteria associated with the soft coral Nephthea sp., and to isolate natural products from these actinobacteria under the guidance of PCR screening for polyketides synthase (PKS) genes. Eleven selective media were used to isolate actinobacteria associated with the soft coral Nephthea sp. collected from Yongxin Island. The isolated actinobacteria were classified on the basis of phylogenetic tree analysis of their 16S rRNA genes. Degenerated primers targeted on conserved KS (ketoacyl-synthase) domain of type I PKS genes were used to screen for potential isolates. The positive isolates were cultured in three different media to check their producing profiles. One bioactive strain that is rich in metabolites was subjected to larger scale fermentation for isolating bioactive natural products. A total of 20 strains were isolated from Nephthea sp., and were categorized into 3 genera including Streptomyces, Dietzia and Salinospora, among which 18 strains were positive in screening with type I PKS genes. Two bioactive compounds rifamycin S and rifamycin W were isolated and identified from Salinospora arenicola SH04. This is the first report of isolating indigenous marine actinobacteria Salinospora from the soft coral Nephthea sp. It provides an example of isolating bioactive secondary metabolites from cultivable actinobacteria associated with Nephthea sp. by PCR screening.

Structure-based mutational analysis of the 4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrier protein recognition and reaction mechanism.

PubMed

Mofid, Mohammad Reza; Finking, Robert; Essen, Lars Oliver; Marahiel, Mohamed A

2004-04-13

The activation of apo-peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases (NRPSs), apo-acyl carrier proteins (ACPs) of polyketide synthases (PKSs), and fatty acid synthases (FASs) to their active holo form is accomplished with dedicated 4'-phosphopantetheinyl transferases (PPTases). They catalyze the transfer of the essential prosthetic group 4'-phosphopantetheine (4'-Ppant) from coenzyme A (CoA) to a highly conserved serine residue in all PCPs and ACPs. PPTases, based on sequence and substrate specifity, have been classified into three types: bacterial holo-acyl carrier protein synthase (AcpS), fatty acid synthase of eukaryotes (FAS2) and Sfp, a PPTase of secondary metabolism. The recently solved crystal structures of AcpS and Sfp-type PPTases with CoA revealed a common alpha + beta-fold with a beta(1)alpha(3)beta(2) motif and similarities in CoA binding and polymerization mode. However, it was not possible to discern neither the PCP binding region of Sfp nor the priming reaction mechanism from the Sfp-CoA cocrystal. In this work, we provide a model for the reaction mechanism based on mutational analysis of Sfp that suggests a reaction mechanism in which the highly conserved E151 deprotonates the hydroxyl group of the invariant serine of PCP. That, in turn, acts as a nucleophile to attack the beta-phosphate of CoA. The Sfp mutants K112, E117, and K120 further revealed that the loop region between beta4 and alpha5 (residues T111-S124) in Sfp is the PCP binding region. Also, residues T44, K75, S89, H90, D107, E109, E151, and K155 that have been shown in the Sfp-CoA cocrystal structure to coordinate CoA are now all confirmed by mutational and biochemical analysis.

Biosynthesis of antimycins with a reconstituted 3-formamidosalicylate pharmacophore in Escherichia coli.

PubMed

Liu, Joyce; Zhu, Xuejun; Seipke, Ryan F; Zhang, Wenjun

2015-05-15

Antimycins are a family of natural products generated from a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line. Although they possess an array of useful biological activities, their structural complexity makes chemical synthesis challenging, and their biosynthesis has thus far been dependent on slow-growing source organisms. Here, we reconstituted the biosynthesis of antimycins in Escherichia coli, a versatile host that is robust and easy to manipulate genetically. Along with Streptomyces genetic studies, the heterologous expression of different combinations of ant genes enabled us to systematically confirm the functions of the modification enzymes, AntHIJKL and AntO, in the biosynthesis of the 3-formamidosalicylate pharmacophore of antimycins. Our E. coli-based antimycin production system can not only be used to engineer the increased production of these bioactive compounds, but it also paves the way for the facile generation of novel and diverse antimycin analogues through combinatorial biosynthesis.

Alkynyl-Containing Peptides of Marine Origin: A Review

PubMed Central

Chai, Qiu-Ye; Yang, Zhen; Lin, Hou-Wen; Han, Bing-Nan

2016-01-01

Since the 1990s, a number of terminal alkynyl residue-containing cyclic/acyclic peptides have been identified from marine organisms, especially cyanobacteria and marine mollusks. This review has presented 66 peptides, which covers over 90% marine peptides with terminal alkynyl fatty acyl units. In fact, more than 90% of these peptides described in the literature are of cyanobacterial origin. Interestingly, all the linear peptides featured with terminal alkyne were solely discovered from marine cyanobacteria. The objective of this article is to provide an overview on the types, structural characterization of these unusual terminal alkynyl fatty acyl units, as well as the sources and biological functions of their composed peptides. Many of these peptides have a variety of biological activities, including antitumor, antibacterial, antimalarial, etc. Further, we have also discussed the evident biosynthetic origin responsible for formation of terminal alkynes of natural PKS (polyketide synthase)/NRPS (nonribosome peptide synthetase) hybrids. PMID:27886049

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.

Comparative genomics of Beauveria bassiana: uncovering signatures of virulence against mosquitoes.

PubMed

Valero-Jiménez, Claudio A; Faino, Luigi; Spring In't Veld, Daphne; Smit, Sandra; Zwaan, Bas J; van Kan, Jan A L

2016-12-01

Entomopathogenic fungi such as Beauveria bassiana are promising biological agents for control of malaria mosquitoes. Indeed, infection with B. bassiana reduces the lifespan of mosquitoes in the laboratory and in the field. Natural isolates of B. bassiana show up to 10-fold differences in virulence between the most and the least virulent isolate. In this study, we sequenced the genomes of five isolates representing the extremes of low/high virulence and three RNA libraries, and applied a genome comparison approach to uncover genetic mechanisms underpinning virulence. A high-quality, near-complete genome assembly was achieved for the highly virulent isolate Bb8028, which was compared to the assemblies of the four other isolates. Whole genome analysis showed a high level of genetic diversity between the five isolates (2.85-16.8 SNPs/kb), which grouped into two distinct phylogenetic clusters. Mating type gene analysis revealed the presence of either the MAT1-1-1 or the MAT1-2-1 gene. Moreover, a putative new MAT gene (MAT1-2-8) was detected in the MAT1-2 locus. Comparative genome analysis revealed that Bb8028 contains 163 genes exclusive for this isolate. These unique genes have a tendency to cluster in the genome and to be often located near the telomeres. Among the genes unique to Bb8028 are a Non-Ribosomal Peptide Synthetase (NRPS) secondary metabolite gene cluster, a polyketide synthase (PKS) gene, and five genes with homology to bacterial toxins. A survey of candidate virulence genes for B. bassiana is presented. Our results indicate several genes and molecular processes that may underpin virulence towards mosquitoes. Thus, the genome sequences of five isolates of B. bassiana provide a better understanding of the natural variation in virulence and will offer a major resource for future research on this important biological control agent.

Characterization of Three Tailoring Enzymes in Dutomycin Biosynthesis and Generation of a Potent Antibacterial Analogue.

PubMed

Sun, Lei; Wang, Siyuan; Zhang, Shuwei; Shao, Lei; Zhang, Qian; Skidmore, Chad; Chang, Cheng-Wei Tom; Yu, Dayu; Zhan, Jixun

2016-07-15

The anthracycline natural product dutomycin and its precursor POK-MD1 were isolated from Streptomyces minoensis NRRL B-5482. The dutomycin biosynthetic gene cluster was identified by genome sequencing and disruption of the ketosynthase gene. Two polyketide synthase (PKS) systems are present in the gene cluster, including a type II PKS and a rare highly reducing iterative type I PKS. The type I PKS DutG repeatedly uses its active sites to create a nine-carbon triketide chain that is subsequently transferred to the α-l-axenose moiety of POK-MD1 at 4″-OH to yield dutomycin. Using a heterologous recombination approach, we disrupted a putative methyltransferase gene (dutMT1) and two glycosyltransferase genes (dutGT1 and dutGT2). Analysis of the metabolites of these mutants revealed the functions of these genes and yielded three dutomycin analogues SW140, SW91, and SW75. The major product SW91 in Streptomyces minoensis NRRL B-5482-ΔDutMT1 was identified as 12-desmethyl-dutomycin, suggesting that DutMT1 is the dedicated 12-methyltransferase. This was confirmed by the in vitro enzymatic assay. DutGT1 and DutGT2 were found to be responsible for the introduction of β-d-amicetose and α-l-axenose, respectively. Dutomycin and SW91 showed strong antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus, whereas POK-MD1 and SW75 had no obvious inhibition, which revealed the essential role of the C-4″ triketide chain in antibacterial activity. The minimal inhibitory concentration of SW91 against the two strains was 0.125 μg mL(-1), lower than that of dutomycin (0.25 μg mL(-1)), indicating that the antibacterial activity of dutomycin can be improved through biosynthetic structural modification.

A functional genomics investigation of allelochemical biosynthesis in Sorghum bicolor root hairs.

PubMed

Baerson, Scott R; Dayan, Franck E; Rimando, Agnes M; Nanayakkara, N P Dhammika; Liu, Chang-Jun; Schröder, Joachim; Fishbein, Mark; Pan, Zhiqiang; Kagan, Isabelle A; Pratt, Lee H; Cordonnier-Pratt, Marie-Michèle; Duke, Stephen O

2008-02-08

Sorghum is considered to be one of the more allelopathic crop species, producing phytotoxins such as the potent benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(Z,Z)-8',11',14'-pentadecatriene]-p-benzoquinone) and its analogs. Sorgoleone likely accounts for much of the allelopathy of Sorghum spp., typically representing the predominant constituent of Sorghum bicolor root exudates. Previous and ongoing studies suggest that the biosynthetic pathway for this plant growth inhibitor occurs in root hair cells, involving a polyketide synthase activity that utilizes an atypical 16:3 fatty acyl-CoA starter unit, resulting in the formation of a pentadecatrienyl resorcinol intermediate. Subsequent modifications of this resorcinolic intermediate are likely to be mediated by S-adenosylmethionine-dependent O-methyltransferases and dihydroxylation by cytochrome P450 monooxygenases, although the precise sequence of reactions has not been determined previously. Analyses performed by gas chromatography-mass spectrometry with sorghum root extracts identified a 3-methyl ether derivative of the likely pentadecatrienyl resorcinol intermediate, indicating that dihydroxylation of the resorcinol ring is preceded by O-methylation at the 3'-position by a novel 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase activity. An expressed sequence tag data set consisting of 5,468 sequences selected at random from an S. bicolor root hair-specific cDNA library was generated to identify candidate sequences potentially encoding enzymes involved in the sorgoleone biosynthetic pathway. Quantitative real time reverse transcription-PCR and recombinant enzyme studies with putative O-methyltransferase sequences obtained from the expressed sequence tag data set have led to the identification of a novel O-methyltransferase highly and predominantly expressed in root hairs (designated SbOMT3), which preferentially utilizes alk(en)ylresorcinols among a panel of benzene-derivative substrates tested. SbOMT3 is therefore proposed to be involved in the biosynthesis of the allelochemical sorgoleone.

Acyl-Homoserine Lactone Production in Nitrifying Bacteria of the Genera Nitrosospira, Nitrobacter, and Nitrospira Identified via a Survey of Putative Quorum-Sensing Genes.

PubMed

Mellbye, Brett L; Spieck, Eva; Bottomley, Peter J; Sayavedra-Soto, Luis A

2017-11-15

The genomes of many bacteria that participate in nitrogen cycling through the process of nitrification contain putative genes associated with acyl-homoserine lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a method of bacterial communication and gene regulation and may be involved in nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. Here, we carried out a broad survey of AHL production in nitrifying bacteria in three steps. First, we analyzed the evolutionary history of AHL synthase and AHL receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus , Nitrobacter , and Nitrospira Next, we screened cultures of both AOB and NOB with uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a bioassay. Our results suggest that an AHL synthase gene is required for, but does not guarantee, cell density-dependent AHL production under the conditions tested. Finally, we utilized mass spectrometry to identify the AHLs produced by the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB Nitrobacter vulgaris and Nitrospira moscoviensis as N -decanoyl-l-homoserine lactone (C 10 -HSL), N -3-hydroxy-tetradecanoyl-l-homoserine lactone (3-OH-C 14 -HSL), a monounsaturated AHL (C 10:1 -HSL), and N -octanoyl-l-homoserine lactone (C 8 -HSL), respectively. Our survey expands the list of AHL-producing nitrifiers to include a representative of Nitrospira lineage II and suggests that AHL production is widespread in nitrifying bacteria. IMPORTANCE Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by nitrifying microorganisms, plays an important role in environmental nitrogen cycling from agricultural fertilization to wastewater treatment. The genomes of many nitrifying bacteria contain genes associated with bacterial cell-cell signaling or quorum sensing (QS). QS is a method of bacterial communication and gene regulation that is well studied in bacterial pathogens, but less is known about QS in environmental systems. Our previous work suggested that QS might be involved in the regulation of nitrogen oxide gas production during nitrite metabolism. This study characterized putative QS signals produced by different genera and species of nitrifiers. Our work lays the foundation for future experiments investigating communication between nitrifying bacteria, the purpose of QS in these microorganisms, and the manipulation of QS during nitrification. Copyright © 2017 American Society for Microbiology.

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

Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis

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

Annaval, Thibault; Rudolf, Jeffrey D.; Chang, Chin-Yuan

Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Though the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. Here, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesismore » of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, our findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis.« less

Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis

DOE PAGES

Annaval, Thibault; Rudolf, Jeffrey D.; Chang, Chin-Yuan; ...

2017-08-30

Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Though the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. Here, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesismore » of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, our findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis.« less

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

Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes’ Expression and Ultrastructural Hyphal Morphologies

PubMed Central

Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

2013-01-01

The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes’ expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis. PMID:24312197

Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis▿

PubMed Central

Fernandes, Chantal; Mendes, Vitor; Costa, Joana; Empadinhas, Nuno; Jorge, Carla; Lamosa, Pedro; Santos, Helena; da Costa, Milton S.

2010-01-01

The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis. PMID:20061481

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite.

PubMed

Masetti, Massimo; Locci, Teresa; Cecchettini, Antonella; Lucchesi, Paolo; Magi, Marta; Malvaldi, Gino; Bruschi, Fabrizio

2004-05-01

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.

Genomics-driven discovery of the pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis

PubMed Central

2013-01-01

Background The antifungal therapy caspofungin is a semi-synthetic derivative of pneumocandin B0, a lipohexapeptide produced by the fungus Glarea lozoyensis, and was the first member of the echinocandin class approved for human therapy. The nonribosomal peptide synthetase (NRPS)-polyketide synthases (PKS) gene cluster responsible for pneumocandin biosynthesis from G. lozoyensis has not been elucidated to date. In this study, we report the elucidation of the pneumocandin biosynthetic gene cluster by whole genome sequencing of the G. lozoyensis wild-type strain ATCC 20868. Results The pneumocandin biosynthetic gene cluster contains a NRPS (GLNRPS4) and a PKS (GLPKS4) arranged in tandem, two cytochrome P450 monooxygenases, seven other modifying enzymes, and genes for L-homotyrosine biosynthesis, a component of the peptide core. Thus, the pneumocandin biosynthetic gene cluster is significantly more autonomous and organized than that of the recently characterized echinocandin B gene cluster. Disruption mutants of GLNRPS4 and GLPKS4 no longer produced the pneumocandins (A0 and B0), and the Δglnrps4 and Δglpks4 mutants lost antifungal activity against the human pathogenic fungus Candida albicans. In addition to pneumocandins, the G. lozoyensis genome encodes a rich repertoire of natural product-encoding genes including 24 PKSs, six NRPSs, five PKS-NRPS hybrids, two dimethylallyl tryptophan synthases, and 14 terpene synthases. Conclusions Characterization of the gene cluster provides a blueprint for engineering new pneumocandin derivatives with improved pharmacological properties. Whole genome estimation of the secondary metabolite-encoding genes from G. lozoyensis provides yet another example of the huge potential for drug discovery from natural products from the fungal kingdom. PMID:23688303

Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations

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

Gokulan, Kuppan; Aggarwal, Anup; Shipman, Lance

2011-07-01

Bacterial acyl carrier protein synthase plays an essential role in the synthesis of fatty acids, nonribosomal peptides and polyketides. In Mycobacterium tuberculosis, AcpS or group I phosphopentatheine transferase exhibits two different structural conformations depending upon the pH. The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS–ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the α2 helix andmore » in the conformation of the α3–α4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4–6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C. ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS–ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS–ADP adopt different conformations depending upon the pH conditions of the crystallization solution.« less

Alkylresorcinol synthases expressed in Sorghum bicolor root hairs play an essential role in the biosynthesis of the allelopathic benzoquinone sorgoleone.

PubMed

Cook, Daniel; Rimando, Agnes M; Clemente, Thomas E; Schröder, Joachim; Dayan, Franck E; Nanayakkara, N P Dhammika; Pan, Zhiqiang; Noonan, Brice P; Fishbein, Mark; Abe, Ikuro; Duke, Stephen O; Baerson, Scott R

2010-03-01

Sorghum bicolor is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone, which likely accounts for many of the allelopathic properties of Sorghum spp. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells and involves the production of an alkylresorcinolic intermediate (5-[(Z,Z)-8',11',14'-pentadecatrienyl]resorcinol) derived from an unusual 16:3Delta(9,12,15) fatty acyl-CoA starter unit. This led to the suggestion of the involvement of one or more alkylresorcinol synthases (ARSs), type III polyketide synthases (PKSs) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. In an effort to characterize the enzymes responsible for the biosynthesis of the pentadecyl resorcinol intermediate, a previously described expressed sequence tag database prepared from isolated S. bicolor (genotype BTx623) root hairs was first mined for all PKS-like sequences. Quantitative real-time RT-PCR analyses revealed that three of these sequences were preferentially expressed in root hairs, two of which (designated ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units in recombinant enzyme studies. Furthermore, RNA interference experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 are likely to participate in the biosynthesis of sorgoleone in planta. The sequences of ARS1 and ARS2 were also used to identify several rice (Oryza sativa) genes encoding ARSs, which are likely involved in the production of defense-related alkylresorcinols.

Alkylresorcinol Synthases Expressed in Sorghum bicolor Root Hairs Play an Essential Role in the Biosynthesis of the Allelopathic Benzoquinone Sorgoleone[W][OA

PubMed Central

Cook, Daniel; Rimando, Agnes M.; Clemente, Thomas E.; Schröder, Joachim; Dayan, Franck E.; Nanayakkara, N.P. Dhammika; Pan, Zhiqiang; Noonan, Brice P.; Fishbein, Mark; Abe, Ikuro; Duke, Stephen O.; Baerson, Scott R.

2010-01-01

Sorghum bicolor is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone, which likely accounts for many of the allelopathic properties of Sorghum spp. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells and involves the production of an alkylresorcinolic intermediate (5-[(Z,Z)-8′,11′,14′-pentadecatrienyl]resorcinol) derived from an unusual 16:3Δ9,12,15 fatty acyl-CoA starter unit. This led to the suggestion of the involvement of one or more alkylresorcinol synthases (ARSs), type III polyketide synthases (PKSs) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. In an effort to characterize the enzymes responsible for the biosynthesis of the pentadecyl resorcinol intermediate, a previously described expressed sequence tag database prepared from isolated S. bicolor (genotype BTx623) root hairs was first mined for all PKS-like sequences. Quantitative real-time RT-PCR analyses revealed that three of these sequences were preferentially expressed in root hairs, two of which (designated ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units in recombinant enzyme studies. Furthermore, RNA interference experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 are likely to participate in the biosynthesis of sorgoleone in planta. The sequences of ARS1 and ARS2 were also used to identify several rice (Oryza sativa) genes encoding ARSs, which are likely involved in the production of defense-related alkylresorcinols. PMID:20348430

Transcriptional activation of a geranylgeranyl diphosphate synthase gene, GGPPS2, isolated from Scoparia dulcis by treatment with methyl jasmonate and yeast extract.

PubMed

Yamamura, Y; Mizuguchi, Y; Taura, F; Kurosaki, F

2014-10-01

A cDNA clone, designated SdGGPPS2, was isolated from young seedlings of Scoparia dulcis. The putative amino acid sequence of the translate of the gene showed high homology with geranylgeranyl diphosphate synthase (GGPPS) from various plant sources, and the N-terminal residues exhibited the characteristics of chloroplast targeting sequence. An appreciable increase in the transcriptional level of SdGGPPS2 was observed by exposure of the leaf tissues of S. dulcis to methyl jasmonate, yeast extract or Ca(2+) ionophore A23187. In contrast, SdGGPPS1, a homologous GGPPS gene of the plant, showed no or only negligible change in the expression level upon treatment with these stimuli. The truncated protein heterologously expressed in Escherichia coli in which the putative targeting domain was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to liberate geranylgeranyl diphosphate. These results suggested that SdGGPPS2 plays physiological roles in methyl jasmonate and yeast extract-induced metabolism in the chloroplast of S. dulcis cells.

Motif-independent prediction of a secondary metabolism gene cluster using comparative genomics: application to sequenced genomes of Aspergillus and ten other filamentous fungal species.

PubMed

Takeda, Itaru; Umemura, Myco; Koike, Hideaki; Asai, Kiyoshi; Machida, Masayuki

2014-08-01

Despite their biological importance, a significant number of genes for secondary metabolite biosynthesis (SMB) remain undetected due largely to the fact that they are highly diverse and are not expressed under a variety of cultivation conditions. Several software tools including SMURF and antiSMASH have been developed to predict fungal SMB gene clusters by finding core genes encoding polyketide synthase, nonribosomal peptide synthetase and dimethylallyltryptophan synthase as well as several others typically present in the cluster. In this work, we have devised a novel comparative genomics method to identify SMB gene clusters that is independent of motif information of the known SMB genes. The method detects SMB gene clusters by searching for a similar order of genes and their presence in nonsyntenic blocks. With this method, we were able to identify many known SMB gene clusters with the core genes in the genomic sequences of 10 filamentous fungi. Furthermore, we have also detected SMB gene clusters without core genes, including the kojic acid biosynthesis gene cluster of Aspergillus oryzae. By varying the detection parameters of the method, a significant difference in the sequence characteristics was detected between the genes residing inside the clusters and those outside the clusters. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Bioprospection of actinobacteria derived from freshwater sediments for their potential to produce antimicrobial compounds.

PubMed

Zothanpuia; Passari, Ajit Kumar; Leo, Vincent Vineeth; Chandra, Preeti; Kumar, Brijesh; Nayak, Chandra; Hashem, Abeer; Abd Allah, Elsayed Fathi; Alqarawi, Abdulaziz A; Singh, Bhim Pratap

2018-05-05

Actinobacteria from freshwater habitats have been explored less than from other habitats in the search for compounds of pharmaceutical value. This study highlighted the abundance of actinobacteria from freshwater sediments of two rivers and one lake, and the isolates were studied for their ability to produce antimicrobial bioactive compounds. 16S rRNA gene sequencing led to the identification of 84 actinobacterial isolates separated into a common genus (Streptomyces) and eight rare genera (Nocardiopsis, Saccharopolyspora, Rhodococcus, Prauserella, Amycolatopsis, Promicromonospora, Kocuria and Micrococcus). All strains that showed significant inhibition potentials were found against Gram-positive, Gram-negative and yeast pathogens. Further, three biosynthetic genes, polyketide synthases type II (PKS II), nonribosomal peptide synthetases (NRPS) and aminodeoxyisochorismate synthase (phzE), were detected in 38, 71 and 29% of the strains, respectively. Six isolates based on their antimicrobial potentials were selected for the detection and quantification of standard antibiotics using ultra performance liquid chromatography (UPLC-ESI-MS/MS) and volatile organic compounds (VOCs) using gas chromatography mass spectrometry (GC/MS). Four antibiotics (fluconazole, trimethoprim, ketoconazole and rifampicin) and 35 VOCs were quantified and determined from the methanolic crude extract of six selected Streptomyces strains. Infectious diseases still remain one of the leading causes of death globally and bacterial infections caused millions of deaths annually. Culturable actinobacteria associated with freshwater lake and river sediments has the prospects for the production of bioactive secondary metabolites.

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

PubMed Central

Hovde, Blake T.; Deodato, Chloe R.; Hunsperger, Heather M.; Ryken, Scott A.; Yost, Will; Jha, Ramesh K.; Patterson, Johnathan; Monnat, Raymond J.; Barlow, Steven B.; Starkenburg, Shawn R.; Cattolico, Rose Ann

2015-01-01

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes. PMID:26397803

Metagenomic Analysis of Upwelling-Affected Brazilian Coastal Seawater Reveals Sequence Domains of Type I PKS and Modular NRPS

PubMed Central

Cuadrat, Rafael R. C.; Cury, Juliano C.; Dávila, Alberto M. R.

2015-01-01

Marine environments harbor a wide range of microorganisms from the three domains of life. These microorganisms have great potential to enable discovery of new enzymes and bioactive compounds for industrial use. However, only ~1% of microorganisms from the environment can currently be identified through cultured isolates, limiting the discovery of new compounds. To overcome this limitation, a metagenomics approach has been widely adopted for biodiversity studies on samples from marine environments. In this study, we screened metagenomes in order to estimate the potential for new natural compound synthesis mediated by diversity in the Polyketide Synthase (PKS) and Nonribosomal Peptide Synthetase (NRPS) genes. The samples were collected from the Praia dos Anjos (Angel’s Beach) surface water—Arraial do Cabo (Rio de Janeiro state, Brazil), an environment affected by upwelling. In order to evaluate the potential for screening natural products in Arraial do Cabo samples, we used KS (keto-synthase) and C (condensation) domains (from PKS and NRPS, respectively) to build Hidden Markov Models (HMM) models. From both samples, a total of 84 KS and 46 C novel domain sequences were obtained, showing the potential of this environment for the discovery of new genes of biotechnological interest. These domains were classified by phylogenetic analysis and this was the first study conducted to screen PKS and NRPS genes in an upwelling affected sample PMID:26633360

The Mitochondrial Permeability Transition Pore: Channel Formation by F-ATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology

PubMed Central

Bernardi, Paolo; Rasola, Andrea; Forte, Michael; Lippe, Giovanna

2015-01-01

The mitochondrial permeability transition (PT) is a permeability increase of the inner mitochondrial membrane mediated by a channel, the permeability transition pore (PTP). After a brief historical introduction, we cover the key regulatory features of the PTP and provide a critical assessment of putative protein components that have been tested by genetic analysis. The discovery that under conditions of oxidative stress the F-ATP synthases of mammals, yeast, and Drosophila can be turned into Ca2+-dependent channels, whose electrophysiological properties match those of the corresponding PTPs, opens new perspectives to the field. We discuss structural and functional features of F-ATP synthases that may provide clues to its transition from an energy-conserving into an energy-dissipating device as well as recent advances on signal transduction to the PTP and on its role in cellular pathophysiology. PMID:26269524

Functional Identification of Valerena-1,10-diene Synthase, a Terpene Synthase Catalyzing a Unique Chemical Cascade in the Biosynthesis of Biologically Active Sesquiterpenes in Valeriana officinalis*

PubMed Central

Yeo, Yun-Soo; Nybo, S. Eric; Chittiboyina, Amar G.; Weerasooriya, Aruna D.; Wang, Yan-Hong; Góngora-Castillo, Elsa; Vaillancourt, Brieanne; Buell, C. Robin; DellaPenna, Dean; Celiz, Mary Dawn; Jones, A. Daniel; Wurtele, Eve Syrkin; Ransom, Nick; Dudareva, Natalia; Shaaban, Khaled A.; Tibrewal, Nidhi; Chandra, Suman; Smillie, Troy; Khan, Ikhlas A.; Coates, Robert M.; Watt, David S.; Chappell, Joe

2013-01-01

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [13C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes. PMID:23243312

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

PubMed

Yeo, Yun-Soo; Nybo, S Eric; Chittiboyina, Amar G; Weerasooriya, Aruna D; Wang, Yan-Hong; Góngora-Castillo, Elsa; Vaillancourt, Brieanne; Buell, C Robin; DellaPenna, Dean; Celiz, Mary Dawn; Jones, A Daniel; Wurtele, Eve Syrkin; Ransom, Nick; Dudareva, Natalia; Shaaban, Khaled A; Tibrewal, Nidhi; Chandra, Suman; Smillie, Troy; Khan, Ikhlas A; Coates, Robert M; Watt, David S; Chappell, Joe

2013-02-01

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

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.

Structure-based virtual screening of hypothetical inhibitors of the enzyme longiborneol synthase-a potential target to reduce Fusarium head blight disease.

PubMed

Bresso, E; Leroux, V; Urban, M; Hammond-Kosack, K E; Maigret, B; Martins, N F

2016-07-01

Fusarium head blight (FHB) is one of the most destructive diseases of wheat and other cereals worldwide. During infection, the Fusarium fungi produce mycotoxins that represent a high risk to human and animal health. Developing small-molecule inhibitors to specifically reduce mycotoxin levels would be highly beneficial since current treatments unspecifically target the Fusarium pathogen. Culmorin possesses a well-known important synergistically virulence role among mycotoxins, and longiborneol synthase appears to be a key enzyme for its synthesis, thus making longiborneol synthase a particularly interesting target. This study aims to discover potent and less toxic agrochemicals against FHB. These compounds would hamper culmorin synthesis by inhibiting longiborneol synthase. In order to select starting molecules for further investigation, we have conducted a structure-based virtual screening investigation. A longiborneol synthase structural model is first built using homology modeling, followed by molecular dynamics simulations that provided the required input for a protein-ligand ensemble docking procedure. From this strategy, the three most interesting compounds (hits) were selected among the 25 top-ranked docked compounds from a library of 15,000 drug-like compounds. These putative inhibitors of longiborneol synthase provide a sound starting point for further studies involving molecular modeling coupled to biochemical experiments. This process could eventually lead to the development of novel approaches to reduce mycotoxin contamination in harvested grain.

Proteoform analysis of lipocalin-type prostaglandin D-synthase from human cerebrospinal fluid by isoelectric focusing and superficially porous liquid chromatography with Fourier transform mass spectrometry.

PubMed

Zhang, Junmei; Corbett, John R; Plymire, Daniel A; Greenberg, Benjamin M; Patrie, Steven M

2014-05-01

Lipocalin-type prostaglandin D-synthase (L-PGDS) in cerebrospinal fluid contributes to the maturation and maintenance of the CNS. L-PGDS PTMs may contribute to pathobiology of different CNS diseases, but methods to monitor its proteoforms are limited. Herein, we combined off-gel IEF and superficially porous LC (SPLC) with Fourier transform MS to characterize common cerebrospinal fluid L-PGDS proteoforms. Across 3D physiochemical space (pI, hydrophobicity, and mass), 217 putative proteoforms were observed from 21 to 24 kDa and pI 5-10. Glycoprotein accurate mass information, combined with MS/MS analysis of peptides generated from 2D-fractionated proteoforms, enabled the putative assignment of 208 proteoforms with varied PTM positional occupants. Fifteen structurally related N-glycans at N29 and N56 were observed, with different N-glycan compositional variants being preferred on each amino acid. We also observed that sialic acid content was a major factor for pI shifts between L-PGDS proteoforms. Other putative PTMs characterized include a core-1 HexHexNAc-O-glycan at S7, acetylation at K16 and K138, sulfonation at S41 and T142, and dioxidation at C43 and C145. The IEF-SPLC-MS platform presented provides 30-40× improved peak capacity versus conventional 2DE and shows potential for repeatable proteoform analysis of surrogate PTM-based biomarkers. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Molecular Control of Polyene Macrolide Biosynthesis

PubMed Central

Santos-Aberturas, Javier; Vicente, Cláudia M.; Guerra, Susana M.; Payero, Tamara D.; Martín, Juan F.; Aparicio, Jesús F.

2011-01-01

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimM. This regulator, which combines an N-terminal PAS domain with a C-terminal helix-turn-helix motif, is highly conserved among polyene biosynthetic gene clusters. PimM, truncated forms of the protein without the PAS domain (PimMΔPAS), and forms containing just the DNA-binding domain (DBD) (PimMDBD) were overexpressed in Escherichia coli as GST-fused proteins. GST-PimM binds directly to eight promoters of the pimaricin cluster, as demonstrated by electrophoretic mobility shift assays. Assays with truncated forms of the protein revealed that the PAS domain does not mediate specificity or the distinct recognition of target genes, which rely on the DBD domain, but significantly reduces binding affinity up to 500-fold. Transcription start points were identified by 5′-rapid amplification of cDNA ends, and the binding regions of PimMDBD were investigated by DNase I protection studies. In all cases, binding took place covering the −35 hexamer box of each promoter, suggesting an interaction of PimM and RNA polymerase to cause transcription activation. Information content analysis of the 16 sequences protected in target promoters was used to deduce the structure of the PimM-binding site. This site displays dyad symmetry, spans 14 nucleotides, and adjusts to the consensus TVGGGAWWTCCCBA. Experimental validation of this binding site was performed by using synthetic DNA duplexes. Binding of PimM to the promoter region of one of the polyketide synthase genes from the Streptomyces nodosus amphotericin cluster containing the consensus binding site was also observed, thus proving the applicability of the findings reported here to other antifungal polyketides. PMID:21187288

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.

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.

Live imaging of β-1,3-glucan synthase FKS-1 in Neurospora crassa hyphae.

PubMed

Sánchez-León, Eddy; Riquelme, Meritxell

2015-09-01

The subcellular localization and dynamics of FKS-1, the putative catalytic subunit of the β-1,3-glucan synthase complex, was analyzed in growing hyphae of Neurospora crassa by live confocal microscopy. GFP-tagged FKS-1 accumulated at the outer layer of the Spitzenkörper (Spk), and at the apical plasma membrane (PM). Fluorescence recovery after photobleaching analysis revealed arrival of FKS-1-containing carriers first at the immediate surroundings of the core region of the Spk, and thereafter to the Spk most outer region. The results obtained here and previous data suggest that FKS-1 is transported to the Spk in macrovesicles. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

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

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

Black perithecial pigmentation in Fusarium species is due to the accumulation of 5-deoxybostrycoidin-based melanin

PubMed Central

Frandsen, Rasmus J. N.; Rasmussen, Silas A.; Knudsen, Peter B.; Uhlig, Silvio; Petersen, Dirk; Lysøe, Erik; Gotfredsen, Charlotte H.; Giese, Henriette; Larsen, Thomas O.

2016-01-01

Biosynthesis of the black perithecial pigment in the filamentous fungus Fusarium graminearum is dependent on the polyketide synthase PGL1 (oPKS3). A seven-membered PGL1 gene cluster was identified by over-expression of the cluster specific transcription factor pglR. Targeted gene replacement showed that PGL1, pglJ, pglM and pglV were essential for the production of the perithecial pigment. Over-expression of PGL1 resulted in the production of 6-O-demethyl-5-deoxybostrycoidin (1), 5-deoxybostrycoidin (2), and three novel compounds 5-deoxybostrycoidin anthrone (3), 6-O-demethyl-5-deoxybostrycoidin anthrone (4) and purpurfusarin (5). The novel dimeric bostrycoidin purpurfusarin (5) was found to inhibit the growth of Candida albicans with an IC50 of 8.0 +/− 1.9 μM. The results show that Fusarium species with black perithecia have a previously undescribed form of 5-deoxybostrycoidin based melanin in their fruiting bodies. PMID:27193384

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

The genome of the social amoeba Dictyostelium discoideum

PubMed Central

Eichinger, L.; Pachebat, J.A.; Glöckner, G.; Rajandream, M.-A.; Sucgang, R.; Berriman, M.; Song, J.; Olsen, R.; Szafranski, K.; Xu, Q.; Tunggal, B.; Kummerfeld, S.; Madera, M.; Konfortov, B. A.; Rivero, F.; Bankier, A. T.; Lehmann, R.; Hamlin, N.; Davies, R.; Gaudet, P.; Fey, P.; Pilcher, K.; Chen, G.; Saunders, D.; Sodergren, E.; Davis, P.; Kerhornou, A.; Nie, X.; Hall, N.; Anjard, C.; Hemphill, L.; Bason, N.; Farbrother, P.; Desany, B.; Just, E.; Morio, T.; Rost, R.; Churcher, C.; Cooper, J.; Haydock, S.; van Driessche, N.; Cronin, A.; Goodhead, I.; Muzny, D.; Mourier, T.; Pain, A.; Lu, M.; Harper, D.; Lindsay, R.; Hauser, H.; James, K.; Quiles, M.; Babu, M. Madan; Saito, T.; Buchrieser, C.; Wardroper, A.; Felder, M.; Thangavelu, M.; Johnson, D.; Knights, A.; Loulseged, H.; Mungall, K.; Oliver, K.; Price, C.; Quail, M.A.; Urushihara, H.; Hernandez, J.; Rabbinowitsch, E.; Steffen, D.; Sanders, M.; Ma, J.; Kohara, Y.; Sharp, S.; Simmonds, M.; Spiegler, S.; Tivey, A.; Sugano, S.; White, B.; Walker, D.; Woodward, J.; Winckler, T.; Tanaka, Y.; Shaulsky, G.; Schleicher, M.; Weinstock, G.; Rosenthal, A.; Cox, E.C.; Chisholm, R. L.; Gibbs, R.; Loomis, W. F.; Platzer, M.; Kay, R. R.; Williams, J.; Dear, P. H.; Noegel, A. A.; Barrell, B.; Kuspa, A.

2005-01-01

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes encode ~12,500 predicted proteins, a high proportion of which have long repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal rDNA element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal/fungal lineage after the plant/animal split, but Dictyostelium appears to have retained more of the diversity of the ancestral genome than either of these two groups. PMID:15875012

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

Discovery and characterization of a marine bacterial SAM-dependent chlorinase

PubMed Central

Eustáquio, Alessandra S; Pojer, Florence; Noel, Joseph P; Moore, Bradley S

2009-01-01

Halogen atom incorporation into a scaffold of bioactive compounds often amplifies biological activity, as is the case for the anticancer agent salinosporamide A (1), a chlorinated natural product from the marine bacterium Salinispora tropica. Significant effort in understanding enzymatic chlorination shows that oxidative routes predominate to form reactive electrophilic or radical chlorine species. Here we report the genetic, biochemical and structural characterization of the chlorinase SalL, which halogenates S-adenosyl-l-methionine (2) with chloride to generate 5′-chloro-5′-deoxyadenosine (3) and l-methionine (4) in a rarely observed nucleophilic substitution strategy analogous to that of Streptomyces cattleya fluorinase. Further metabolic tailoring produces a halogenated polyketide synthase substrate specific for salinosporamide A biosynthesis. SalL also accepts bromide and iodide as substrates, but not fluoride. High-resolution crystal structures of SalL and active site mutants complexed with substrates and products support the SN2 nucleophilic substitution mechanism and further illuminate halide specificity in this newly discovered halogenase family. PMID:18059261

Genetic Insights Into Pyralomicin Biosynthesis in Nonomuraea spiralis IMC A-0156

PubMed Central

Flatt, Patricia M.; Wu, Xiumei; Perry, Steven; Mahmud, Taifo

2013-01-01

The biosynthetic gene cluster for the pyralomicin antibiotics has been cloned and sequenced from Nonomuraea spiralis IMC A-0156. The 41-kb gene cluster contains 27 ORFs predicted to encode all of the functions for pyralomicin biosynthesis. This includes non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) required for the formation of the benzopyranopyrrole core unit, as well as a suite of tailoring enzymes (e.g., four halogenases, an O-methyltransferase, and an N-glycosyltransferase) necessary for further modifications of the core structure. The N-glycosyltransferase is predicted to transfer either glucose or a pseudosugar (cyclitol) to the aglycone. A gene cassette encoding C7-cyclitol biosynthetic enzymes was identified upstream of the benzopyranopyrrole-specific ORFs. Targeted disruption of the gene encoding the N-glycosyltransferase, prlH, abolished pyralomicin production and recombinant expression of PrlA confirms the activity of this enzyme as a sugar phosphate cyclase (SPC) involved in the formation of the C7-cyclitol moiety. PMID:23607523

[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.

Sponge-Derived Kocuria and Micrococcus spp. as Sources of the New Thiazolyl Peptide Antibiotic Kocurin

PubMed Central

Palomo, Sara; González, Ignacio; de la Cruz, Mercedes; Martín, Jesús; Tormo, José Rubén; Anderson, Matthew; Hill, Russell T.; Vicente, Francisca; Reyes, Fernando; Genilloud, Olga

2013-01-01

Forty four marine actinomycetes of the family Microccocaceae isolated from sponges collected primarily in Florida Keys (USA) were selected from our strain collection to be studied as new sources for the production of bioactive natural products. A 16S rRNA gene based phylogenetic analysis showed that the strains are members of the genera Kocuria and Micrococcus. To assess their biosynthetic potential, the strains were PCR screened for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS) and polyketide synthases (PKS). A small extract collection of 528 crude extracts generated from nutritional microfermentation arrays was tested for the production of bioactive secondary metabolites against clinically relevant strains (Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Candida albicans). Three independent isolates were shown to produce a new anti-MRSA bioactive compound that was identified as kocurin, a new member of the thiazolyl peptide family of antibiotics emphasizing the role of this family as a prolific resource for novel drugs. PMID:23538871

Sponge-derived Kocuria and Micrococcus spp. as sources of the new thiazolyl peptide antibiotic kocurin.

PubMed

Palomo, Sara; González, Ignacio; de la Cruz, Mercedes; Martín, Jesús; Tormo, José Rubén; Anderson, Matthew; Hill, Russell T; Vicente, Francisca; Reyes, Fernando; Genilloud, Olga

2013-03-28

Forty four marine actinomycetes of the family Microccocaceae isolated from sponges collected primarily in Florida Keys (USA) were selected from our strain collection to be studied as new sources for the production of bioactive natural products. A 16S rRNA gene based phylogenetic analysis showed that the strains are members of the genera Kocuria and Micrococcus. To assess their biosynthetic potential, the strains were PCR screened for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS) and polyketide synthases (PKS). A small extract collection of 528 crude extracts generated from nutritional microfermentation arrays was tested for the production of bioactive secondary metabolites against clinically relevant strains (Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Candida albicans). Three independent isolates were shown to produce a new anti-MRSA bioactive compound that was identified as kocurin, a new member of the thiazolyl peptide family of antibiotics emphasizing the role of this family as a prolific resource for novel drugs.

Crystallization and x-ray diffraction analysis of a putative bacterial class I labdane-related diterpene synthase [Crysallization and preliminary x-ray diffraction analysis of a bacterial class I labdane-related diterpene synthase

DOE PAGES

Serrano-Posada, Hugo; Centeno-Leija, Sara; Rojas-Trejo, Sonia; ...

2015-08-25

Here, labdane-related diterpenoids are natural products with potential pharmaceutical applications that are rarely found in bacteria. Here, a putative class I labdane-related diterpene synthase (LrdC) identified by genome mining in a streptomycete was successfully crystallized using the microbatch method. Crystals of the LrdC enzyme were obtained in a holo form with its natural cofactor Mg 2+ (LrdC-Mg 2+) and in complex with inorganic pyrophosphate (PP i) (LrdC-Mg 2+–PP i). Crystals of native LrdC-Mg 2+ diffracted to 2.50 Å resolution and belonged to the trigonal space group P3 221, with unit-cell parameters a = b = 107.1, c = 89.2 Å.more » Crystals of the LrdC-Mg 2+–PP i complex grown in the same conditions as the native enzyme with PEG 8000 diffracted to 2.36 Å resolution and also belonged to the trigonal space group P3 221. Crystals of the LrdC-Mg 2+–PP i complex grown in a second crystallization condition with PEG 3350 diffracted to 2.57 Å resolution and belonged to the monoclinic space group P2 1, with unit-cell parameters a = 49.9, b = 104.1, c = 66.5 Å, β = 111.4°. The structure was determined by the single-wavelength anomalous dispersion (SAD) technique using the osmium signal from a potassium hexachloroosmate (IV) derivative.« 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.

Multiple roles of the prostaglandin D2 signaling pathway in reproduction.

PubMed

Rossitto, Moïra; Ujjan, Safdar; Poulat, Francis; Boizet-Bonhoure, Brigitte

2015-01-01

Prostaglandins signaling molecules are involved in numerous physiological processes. They are produced by several enzyme-limited reactions upon fatty acids, which are catalyzed by two cyclooxygenases and prostaglandin synthases. In particular, the prostaglandins E2 (PGE2), D2 (PGD2), and F2 (PGF2 α) have been shown to be involved in female reproductive mechanisms. Furthermore, widespread expression of lipocalin- and hematopoietic-PGD2 synthases in the male reproductive tract supports the purported roles of PGD2 in the development of both embryonic and adult testes, sperm maturation, and spermatogenesis. In this review, we summarize the putative roles of PGD2 signaling and the roles of both PGD2 synthases in testicular formation and function. We review the data reporting the involvement of PGD2 signaling in the differentiation of Sertoli and germ cells of the embryonic testis. Furthermore, we discuss the roles of lipocalin-PGD2 synthase in steroidogenesis and spermatogenesis, in terms of lipid molecule transport and PGD2 production. Finally, we discuss the hypothesis that PGD2 signaling may be affected in certain reproductive diseases, such as infertility, cryptorchidism, and testicular cancer. © 2015 Society for Reproduction and Fertility.

Discovery, biosynthesis, and rational engineering of novel enterocin and wailupemycin polyketide analogues.

PubMed

Kalaitzis, John A

2013-01-01

The marine actinomycete Streptomyces maritimus produces a structurally diverse set of unusual polyketide natural products including the major metabolite enterocin. Investigations of enterocin biosynthesis revealed that the unique carbon skeleton is derived from an aromatic polyketide pathway which is genetically coded by the 21.3 kb enc gene cluster in S. maritimus. Characterization of the enc biosynthesis gene cluster and subsequent manipulation of it via heterologous expression and/or mutagenesis enabled the discovery of other enc-based metabolites that were produced in only very minor amounts in the wild type. Also described are techniques used to harness the enterocin biosynthetic machinery in order to generate unnatural enc-derived polyketide analogues. This review focuses upon the molecular methods used in combination with classical natural products detection and isolation techniques to access minor metabolites of the S. maritimus secondary metabolome.

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.

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

DOE PAGES

Hovde, Blake T.; Deodato, Chloe R.; Hunsperger, Heather M.; ...

2015-09-23

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. Themore » nuclear genome of C. tobin is small (59 Mb), compact (~40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. In conclusion, the Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.« less

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

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

Hovde, Blake T.; Deodato, Chloe R.; Hunsperger, Heather M.

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. Themore » nuclear genome of C. tobin is small (59 Mb), compact (~40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. In conclusion, the Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.« less

[Diversity of cultivable actinobacteria in Xinghu wetland sediments].

PubMed

Xue, Dong; Zhao, Guozhen; Yao, Qing; Zhao, Haiquan; Zhu, Honghui

2015-11-04

To study the diversity of cultivable actinobacteria in Xinghu wetland and screen actinobacteria with a pharmaceutical potential for producing biologically active secondary metabolites. We studied the diversity of actinobacteria isolated from Xinghu wetland by using different selective isolation media and methods. The high bioactive actinobacteria were identified and further investigated for the presence of polyketide synthases (PKS-I, PKS-II), nonribosomal peptide synthetases (NRPS), 3-amino-5-hydroxybenzoic acid synthases (AHBA) and 3-hydroxy-3-methylglutaryl Coenzyme A (HMG CoA) sequences by specific amplification. More than 300 actinobacteria were isolated, and 135 isolates were selected on the basis of their morphologies on different media and were further characterized by 16S rRNA gene sequencing. The isolates belonged to 7 orders, 10 families, 13 genera, Streptomyces was the most frequently isolated genus, followed by the genera Micromonospora and Nocardia. Twenty-four isolates showed high activity against Staphylococcus aureus and Escherichia coli, but there no strain displaying antagonistic activity against Salmonella sp. High frequencies of positive PCR amplification were obtained for PKS-I (16.7%, 4/24), PKS-II (62.5%,15/24), NRPS (16.7%, 4/24), HMG CoA (29.2%, 7/24) and AHBA (12.5%, 3/24) biosynthetic systems. High Performance Liquid Chromatography showed that strain XD7, XD114, XD128 produce lots of secondary metabolites. This study indicated that actinobacteria isolated from Xinghu wetland are abundant and have potentially beneficial and diverse bioactivities which should be pursued for their biotechnical promise.

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.

Evidence for a structural role for acid-fast lipids in oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria.

PubMed

Bushkin, G Guy; Motari, Edwin; Carpentieri, Andrea; Dubey, Jitender P; Costello, Catherine E; Robbins, Phillips W; Samuelson, John

2013-09-03

Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp. cause diarrhea in humans and animals, while Toxoplasma causes disseminated infections in fetuses and untreated AIDS patients. Eimeria is a major pathogen of commercial chickens. Oocysts, which are the infectious form of Cryptosporidium and Eimeria and one of two infectious forms of Toxoplasma (the other is tissue cysts in undercooked meat), have a multilayered wall. Recently we showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria is a porous scaffold of fibers of β-1,3-glucan, which are also present in fungal walls but are absent from Cryptosporidium oocyst walls. Here we present evidence for a structural role for lipids in the oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. Briefly, oocyst walls of each organism label with acid-fast stains that bind to lipids in the walls of mycobacteria. Polyketide synthases similar to those that make mycobacterial wall lipids are abundant in oocysts of Toxoplasma and Eimeria and are predicted in Cryptosporidium. The outer layer of oocyst wall of Eimeria and the entire oocyst wall of Cryptosporidium are dissolved by organic solvents. Oocyst wall lipids are complex mixtures of triglycerides, some of which contain polyhydroxy fatty acyl chains like those present in plant cutin or elongated fatty acyl chains like mycolic acids. We propose a two-layered model of the oocyst wall (glucan and acid-fast lipids) that resembles the two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plants (cellulose and cutin). Oocysts, which are essential for the fecal-oral spread of coccidia, have a wall that is thought responsible for their survival in the environment and for their transit through the stomach and small intestine. While oocyst walls of Toxoplasma and Eimeria are strengthened by a porous scaffold of fibrils of β-1,3-glucan and by proteins cross-linked by dityrosines, both are absent from walls of Cryptosporidium. We show here that all oocyst walls are acid fast, have a rigid bilayer, dissolve in organic solvents, and contain a complex set of triglycerides rich in polyhydroxy and long fatty acyl chains that might be synthesized by an abundant polyketide synthase. These results suggest the possibility that coccidia build a waxy coat of acid-fast lipids in the oocyst wall that makes them resistant to environmental stress.

Transcriptome profiling of the Australian arid-land plant Eremophila serrulata (A.DC.) Druce (Scrophulariaceae) for the identification of monoterpene synthases.

PubMed

Kracht, Octavia Natascha; Ammann, Ann-Christin; Stockmann, Julia; Wibberg, Daniel; Kalinowski, Jörn; Piotrowski, Markus; Kerr, Russell; Brück, Thomas; Kourist, Robert

2017-04-01

Plant terpenoids are a large and highly diverse class of metabolites with an important role in the immune defense. They find wide industrial application as active pharmaceutical ingredients, aroma and fragrance compounds. Several Eremophila sp. derived terpenoids have been documented. To elucidate the terpenoid metabolism, the transcriptome of juvenile and mature Eremophila serrulata (A.DC.) Druce (Scrophulariaceae) leaves was sequenced and a transcript library was generated. We report on the first transcriptomic dataset of an Eremophila plant. IlluminaMiSeq sequencing (2 × 300 bp) revealed 7,093,266 paired reads, which could be assembled to 34,505 isogroups. To enable detection of terpene biosynthetic genes, leaves were separately treated with methyl jasmonate, a well-documented inducer of plant secondary metabolites. In total, 21 putative terpene synthase genes were detected in the transcriptome data. Two terpene synthase isoenzymatic genes, termed ES01 and ES02, were successfully expressed in E. coli. The resulting proteins catalyzed the conversion of geranyl pyrophosphate, the universal substrate of monoterpene synthases to myrcene and Z-(b)-ocimene, respectively. The transcriptomic data and the discovery of the first terpene synthases from Eremophila serrulata are the initial step for the understanding of the terpene metabolism in this medicinally important plant genus. Copyright © 2017 Elsevier Ltd. All rights reserved.

Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity.

PubMed Central

Gojković, Z; Sandrini, M P; Piskur, J

2001-01-01

beta-Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamyl-beta-alanine as the sole nitrogen source and exhibits diminished beta-alanine synthase activity was used to clone analogous genes from different eukaryotes. Putative PYD3 sequences from the yeast S. kluyveri, the slime mold Dictyostelium discoideum, and the fruit fly Drosophila melanogaster complemented the pyd3 defect. When the S. kluyveri PYD3 gene was expressed in S. cerevisiae, which has no pyrimidine catabolic pathway, it enabled growth on N-carbamyl-beta-alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta-alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial N-carbamyl amidohydrolases. All three beta-alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta-alanine, but not by uracil. This work establishes S. kluyveri as a model organism for studying pyrimidine degradation and beta-alanine production in eukaryotes. PMID:11454750

An unusual plant triterpene synthase with predominant α-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica.

PubMed

Brendolise, Cyril; Yauk, Yar-Khing; Eberhard, Ellen D; Wang, Mindy; Chagne, David; Andre, Christelle; Greenwood, David R; Beuning, Lesley L

2011-07-01

The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. α-Amyrin and β-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α-amyrin to β-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α-amyrin produced is > 80% of the total product and is, therefore, primarily an α-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either α-amyrin and/or β-amyrin synthase. MdOSC1 and MdOSC3 clustered with the multifunctional triterpene synthases, whereas MdOSC2 was most similar to the β-amyrin synthases. © 2011 The New Zealand Institute for Plant and Food Research Limited. Journal compilation © 2011 FEBS.

Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei

PubMed Central

Derntl, Christian; Rassinger, Alice; Srebotnik, Ewald; Mach, Robert L.

2016-01-01

ABSTRACT The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum. In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1. IMPORTANCE Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum. The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism. PMID:27520818

An Insight into the Secondary Metabolism of Muscodor yucatanensis: Small-Molecule Epigenetic Modifiers Induce Expression of Secondary Metabolism-Related Genes and Production of New Metabolites in the Endophyte.

PubMed

Qadri, Masroor; Nalli, Yedukondalu; Jain, Shreyans K; Chaubey, Asha; Ali, Asif; Strobel, Gary A; Vishwakarma, Ram A; Riyaz-Ul-Hassan, Syed

2017-05-01

Muscodor spp. are proficient producers of bioactive volatile organic compounds (VOCs) with many potential applications. However, all members of this genus produce varying amounts and types of VOCs which suggests the involvement of epigenetics as a possible explanation. The members of this genus are poorly explored for the production of soluble compounds (extrolites). In this study, the polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes from an endophyte, Muscodor yucatanensis Ni30, were cloned and sequenced. The PKS genes belonged to reduced, partially reduced, non-reduced, and highly reduced subtypes. Strains over-expressing PKS genes were developed through the use of small-molecule epigenetic modifiers (suberoylanilide hydroxamic acid (SAHA) and 5-azacytidine). The putative epigenetic variants of this organism differed considerably from the wild type in morphological features and cultural characteristics as well as metabolites that were produced. Each variant produced a different set of VOCs distinct from the wild type, and several VOCs including methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)hexane-2,4-diol and 2-carboxymethyl-3-n-hexylmaleic appeared in the variant strains, the production of which could be attributed to the activity of otherwise silent PKS genes. The bioactive extrolite brefeldin A was isolated and characterized from the wild type. However, this metabolite was not detected in EV-1, but instead, two other products were isolated and characterized as ergosterol and xylaguaianol C. Hence, M. yucatanensis has the genetic potential to produce several previously undetectable VOCs and organic solvent soluble products. It is also the case that small-molecule epigenetic modifiers can be used to produce stable variant strains of fungi with the potential to produce new molecules. Finally, this work hints to the prospect that the epigenetics of an endophytic microorganism can be influenced by any number of environmental and chemical factors associated with its host plant which may help to explain the enormous chemical diversity of secondary metabolic products found in Muscodor spp.

A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

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

Shi, Dashuang; Li, Yongdong; Cabrera-Luque, Juan

2012-05-24

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 {angstrom} resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS aremore » tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26{sup o} is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.« less

Modulation of medium-chain fatty acid synthesis in Synechococcus sp. PCC 7002 by replacing FabH with a Chaetoceros Ketoacyl-ACP synthase

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

Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novomore » assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase Ill increased MCFA synthesis up to fivefold. In conclusion, the level of increase is dependent on promoter strength and culturing conditions.« less

Modulation of medium-chain fatty acid synthesis in Synechococcus sp. PCC 7002 by replacing FabH with a Chaetoceros Ketoacyl-ACP synthase

DOE PAGES

Gu, Huiya; Jinkerson, Robert E.; Davies, Fiona K.; ...

2016-05-26

The isolation or engineering of algal cells synthesizing high levels of medium-chain fatty acids (MCFAs) is attractive to mitigate the high clouding point of longer chain fatty acids in algal based biodiesel. To develop a more informed understanding of MCFA synthesis in photosynthetic microorganisms, we isolated several algae from Great Salt Lake and screened this collection for MCFA accumulation to identify strains naturally accumulating high levels of MCFA. A diatom, Chaetoceros sp. GSL56, accumulated particularly high levels of C14 (up to 40%), with the majority of C14 fatty acids allocated in triacylglycerols. Using whole cell transcriptome sequencing and de novomore » assembly, putative genes encoding fatty acid synthesis enzymes were identified. Enzymes from this Chaetoceros sp. were expressed in the cyanobacterium Synechococcus sp. PCC 7002 to validate gene function and to determine whether eukaryotic enzymes putatively lacking bacterial evolutionary control mechanisms could be used to improve MCFA production in this promising production strain. Replacement of the Synechococcus 7002 native FabH with a Chaetoceros ketoacyl-ACP synthase Ill increased MCFA synthesis up to fivefold. In conclusion, the level of increase is dependent on promoter strength and culturing conditions.« less

Genetic Screening Strategy for Rapid Access to Polyether Ionophore Producers and Products in Actinomycetes ▿ †

PubMed Central

Wang, Hao; Liu, Ning; Xi, Lijun; Rong, Xiaoying; Ruan, Jisheng; Huang, Ying

2011-01-01

Polyether ionophores are a unique class of polyketides with broad-spectrum activity and outstanding potency for the control of drug-resistant bacteria and parasites, and they are produced exclusively by actinomycetes. A special epoxidase gene encoding a critical tailoring enzyme involved in the biosynthesis of these compounds has been found in all five of the complete gene clusters of polyether ionophores published so far. To detect potential producer strains of these antibiotics, a pair of degenerate primers was designed according to the conserved regions of the five known polyether epoxidases. A total of 44 putative polyether epoxidase gene-positive strains were obtained by the PCR-based screening of 1,068 actinomycetes isolated from eight different habitats and 236 reference strains encompassing eight major families of Actinomycetales. The isolates spanned a wide taxonomic diversity based on 16S rRNA gene analysis, and actinomycetes isolated from acidic soils seemed to be a promising source of polyether ionophores. Four genera were detected to contain putative polyether epoxidases, including Micromonospora, which has not previously been reported to produce polyether ionophores. The designed primers also detected putative epoxidase genes from diverse known producer strains that produce polyether ionophores unrelated to the five published gene clusters. Moreover, phylogenetic and chemical analyses showed a strong correlation between the sequence of polyether epoxidases and the structure of encoded polyethers. Thirteen positive isolates were proven to be polyether ionophore producers as expected, and two new analogues were found. These results demonstrate the feasibility of using this epoxidase gene screening strategy to aid the rapid identification of known products and the discovery of unknown polyethers in actinomycetes. PMID:21421776

Genetic screening strategy for rapid access to polyether ionophore producers and products in actinomycetes.

PubMed

Wang, Hao; Liu, Ning; Xi, Lijun; Rong, Xiaoying; Ruan, Jisheng; Huang, Ying

2011-05-01

Polyether ionophores are a unique class of polyketides with broad-spectrum activity and outstanding potency for the control of drug-resistant bacteria and parasites, and they are produced exclusively by actinomycetes. A special epoxidase gene encoding a critical tailoring enzyme involved in the biosynthesis of these compounds has been found in all five of the complete gene clusters of polyether ionophores published so far. To detect potential producer strains of these antibiotics, a pair of degenerate primers was designed according to the conserved regions of the five known polyether epoxidases. A total of 44 putative polyether epoxidase gene-positive strains were obtained by the PCR-based screening of 1,068 actinomycetes isolated from eight different habitats and 236 reference strains encompassing eight major families of Actinomycetales. The isolates spanned a wide taxonomic diversity based on 16S rRNA gene analysis, and actinomycetes isolated from acidic soils seemed to be a promising source of polyether ionophores. Four genera were detected to contain putative polyether epoxidases, including Micromonospora, which has not previously been reported to produce polyether ionophores. The designed primers also detected putative epoxidase genes from diverse known producer strains that produce polyether ionophores unrelated to the five published gene clusters. Moreover, phylogenetic and chemical analyses showed a strong correlation between the sequence of polyether epoxidases and the structure of encoded polyethers. Thirteen positive isolates were proven to be polyether ionophore producers as expected, and two new analogues were found. These results demonstrate the feasibility of using this epoxidase gene screening strategy to aid the rapid identification of known products and the discovery of unknown polyethers in actinomycetes.

Olive phenolic compounds: metabolic and transcriptional profiling during fruit development

PubMed Central

2012-01-01

Background Olive (Olea europaea L.) fruits contain numerous secondary metabolites, primarily phenolics, terpenes and sterols, some of which are particularly interesting for their nutraceutical properties. This study will attempt to provide further insight into the profile of olive phenolic compounds during fruit development and to identify the major genetic determinants of phenolic metabolism. Results The concentration of the major phenolic compounds, such as oleuropein, demethyloleuropein, 3–4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans, were measured in the developing fruits of 12 olive cultivars. The content of these compounds varied significantly among the cultivars and decreased during fruit development and maturation, with some compounds showing specificity for certain cultivars. Thirty-five olive transcripts homologous to genes involved in the pathways of the main secondary metabolites were identified from the massive sequencing data of the olive fruit transcriptome or from cDNA-AFLP analysis. Their mRNA levels were determined using RT-qPCR analysis on fruits of high- and low-phenolic varieties (Coratina and Dolce d’Andria, respectively) during three different fruit developmental stages. A strong correlation was observed between phenolic compound concentrations and transcripts putatively involved in their biosynthesis, suggesting a transcriptional regulation of the corresponding pathways. OeDXS, OeGES, OeGE10H and OeADH, encoding putative 1-deoxy-D-xylulose-5-P synthase, geraniol synthase, geraniol 10-hydroxylase and arogenate dehydrogenase, respectively, were almost exclusively present at 45 days after flowering (DAF), suggesting that these compounds might play a key role in regulating secoiridoid accumulation during fruit development. Conclusions Metabolic and transcriptional profiling led to the identification of some major players putatively involved in biosynthesis of secondary compounds in the olive tree. Our data represent the first step towards the functional characterisation of important genes for the determination of olive fruit quality. PMID:22963618

A Putative Histone Deacetylase Modulates the Biosynthesis of Pestalotiollide B and Conidiation in Pestalotiopsis microspora.

PubMed

Niu, Xueliang; Hao, Xiaoran; Hong, Zhangyong; Chen, Longfei; Yu, Xi; Zhu, Xudong

2015-05-01

Fungi of the genus Pestalotiopsis have drawn attention for their capability to produce an array of bioactive secondary metabolites that have potential for drug development. Here, we report the determination of a polyketide derivative compound, pestalotiollide B, in the culture of the saprophytic fungus Pestalotiopsis microspora NK17. Structural information acquired by analyses with a set of spectroscopic and chromatographic techniques suggests that pestalotiollide B has the same skeleton as the penicillide derivatives, dibenzodioxocinones, which are inhibitors of cholesterol ester transfer protein (CETP), and as purpactins A and C', inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Strain NK17 can make a fairly high yield of pestalotiollide B (i.e., up to 7.22 mg/l) in a constitutive manner in liquid culture. Moreover, we found that a putative histone deacetylase gene, designated as hid1, played a role in the biosynthesis of pestalotiollide B. In the hid1 null mutant, the yield of pestalotiollide B increased approximately 2-fold to 15.90 mg/l. In contrast, deletion of gene hid1 led to a dramatic decrease of conidia production of the fungus. These results suggest that hid1 is a modulator, concerting secondary metabolism and development such as conidiation in P. microspora. Our work may help with the investigation into the biosynthesis of pestalotiollide B and the development for new CETP and ACAT inhibitors.

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...

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.

The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.

PubMed

Wirtz, M; Berkowitz, O; Droux, M; Hell, R

2001-02-01

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.

Functional characterization of ent-copalyl diphosphate synthase from Andrographis paniculata with putative involvement in andrographolides biosynthesis.

PubMed

Shen, Qinqin; Li, Lixia; Jiang, Yu; Wang, Qiang

2016-01-01

To characterize the ent-copalyl diphosphate (ent-CPP) synthase involved in the biosynthetic pathway of andrographolides in a medicinal plant, Andrographis paniculata. The ent-CPP synthase (ent-CPS) gene was cloned from A. paniculata and its encoded ApCPS was demonstrated to react with (E,E,E)-geranylgeranyl diphosphate to form ent-CPP through recombinant expression in Escherichia coli. Site-directed mutagenesis of the Asp to Ala in the conserved DXDD motif of ApCPS resulted in loss of function. One Arg is located in the conserved position close to DXDD motif indicating the involvement of ApCPS in specialized metabolism. In addition, RT-PCR analysis revealed that ApCPS was expressed in all tissues of A. paniculata at all growth stages, which is consistent with andrographolides accumulating in these organs. Methyl jasmonate induced ApCPS gene expression, matching inducible accumulation of andrographolides in vivo. ApCPS is the first ent-CPS characterized in A. paniculata and is suggested to be involved in biosynthesis of andrographolides that have high pharmaceutical values.

Isolation of Persicaria minor sesquiterpene synthase promoter and its deletions for transgenic Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Omar, Aimi Farehah; Ismail, Ismanizan

2016-11-01

Sesquiterpene synthase (SS) catalyzes the formation of sesquiterpenes from farnesyl diphosphate (FDP) via carbocation intermediates. In this study, the promoter region of sesquiterpene synthase was isolated from Persicaria minor to identify possible cis-acting elements in the promoter. The full-length PmSS promoter of P. minor is 1824-bp sequences. The sequence was analyzed and several putative cis-acting regulatory elements were identified. Three cis-acting regulatory elements were selected for deletion analysis which are cis-acting element involved in wound responsiveness (WUN), cis - acting element involved in defense and stress responsiveness (TC) and cis-acting element involved in ABA responsiveness (ABRE). Series of deletions were conducted to assess the promoter activity producing three truncated fragments promoter; Prom 2 1606-bp, Prom 3 1144- bp, and Prom 4 921-bp. The full-length promoter and its deletion series were cloned into the pBGWFS7 vector which contain β-glucuronidase (GUS) gene and green fluorescent protein (GFP) as the reporter gene. All constructs were successfully transformed into Arabidopsis thaliana based on PCR of positive BASTA resistance plants.

Genome-wide identification, functional and evolutionary analysis of terpene synthases in pineapple.

PubMed

Chen, Xiaoe; Yang, Wei; Zhang, Liqin; Wu, Xianmiao; Cheng, Tian; Li, Guanglin

2017-10-01

Terpene synthases (TPSs) are vital for the biosynthesis of active terpenoids, which have important physiological, ecological and medicinal value. Although terpenoids have been reported in pineapple (Ananas comosus), genome-wide investigations of the TPS genes responsible for pineapple terpenoid synthesis are still lacking. By integrating pineapple genome and proteome data, twenty-one putative terpene synthase genes were found in pineapple and divided into five subfamilies. Tandem duplication is the cause of TPS gene family duplication. Furthermore, functional differentiation between each TPS subfamily may have occurred for several reasons. Sixty-two key amino acid sites were identified as being type-II functionally divergence between TPS-a and TPS-c subfamily. Finally, coevolution analysis indicated that multiple amino acid residues are involved in coevolutionary processes. In addition, the enzyme activity of two TPSs were tested. This genome-wide identification, functional and evolutionary analysis of pineapple TPS genes provide a new insight into understanding the roles of TPS family and lay the basis for further characterizing the function and evolution of TPS gene family. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of a novel 8R,11S-linoleate diol synthase from Penicillium chrysogenum by identification of its enzymatic products[S

PubMed Central

Shin, Kyung-Chul; Seo, Min-Ju; Oh, Deok-Kun

2016-01-01

To identify novel fatty acid diol synthases, putative candidate sequences from Penicillium species were analyzed, and hydroxy fatty acid production by crude Penicillium enzyme extracts was assessed. Penicillium chrysogenum was found to produce an unknown dihydroxy fatty acid, a candidate gene implicated in this production was cloned and expressed, and the expressed enzyme was purified. The product obtained by the reaction of the purified enzyme with linoleic acid was identified as 8R,11S-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8R,11S-DiHODE). The catalytic efficiency of this enzyme toward linoleic acid was the highest among the unsaturated fatty acids tested, indicating that this enzyme was a novel 8R,11S-linoleate diol synthase (8R,11S-LDS). A sexual stage in the life cycle of P. chrysogenum has recently been discovered, and 8R,11S-DiHODE produced by 8R,11S-LDS may constitute a precocious sexual inducer factor, responsible for regulating the sexual and asexual cycles of this fungus. PMID:26681780

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.

Draft Genome Sequence of Janthinobacterium sp. Strain ROICE36, a Putative Secondary Metabolite-Synthesizing Bacterium Isolated from Antarctic Snow

PubMed Central

Chiriac, Cecilia; Baricz, Andreea

2018-01-01

ABSTRACT The draft genome assembly of Janthinobacterium sp. strain ROICE36 has 207 contigs, with a total genome size of 5,977,006 bp and a G+C content of 62%. Preliminary genome analysis identified 5,363 protein-coding genes and a total of 7 secondary metabolic gene clusters (encoding bacteriocins, nonribosomal peptide-synthetase [NRPS], terpene, hserlactone, and other ketide synthases). PMID:29650588

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...

Characterization of Cyanobacterial Hydrocarbon Composition and Distribution of Biosynthetic Pathways

PubMed Central

Coates, R. Cameron; Podell, Sheila; Korobeynikov, Anton; Lapidus, Alla; Pevzner, Pavel; Sherman, David H.; Allen, Eric E.; Gerwick, Lena; Gerwick, William H.

2014-01-01

Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene) and methyl group positions (3-, 4- and 5-methylheptadecane) for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO). The second involves a polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS). Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both. PMID:24475038

Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the Streptomyces ambofaciens Linear Chromosome

PubMed Central

Pang, Xiuhua; Aigle, Bertrand; Girardet, Jean-Michel; Mangenot, Sophie; Pernodet, Jean-Luc; Decaris, Bernard; Leblond, Pierre

2004-01-01

Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the β-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production. PMID:14742212

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

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.

Insect-Induced Conifer Defense. White Pine Weevil and Methyl Jasmonate Induce Traumatic Resinosis, de Novo Formed Volatile Emissions, and Accumulation of Terpenoid Synthase and Putative Octadecanoid Pathway Transcripts in Sitka Spruce1[w

PubMed Central

Miller, Barbara; Madilao, Lufiani L.; Ralph, Steven; Bohlmann, Jörg

2005-01-01

Stem-boring insects and methyl jasmonate (MeJA) are thought to induce similar complex chemical and anatomical defenses in conifers. To compare insect- and MeJA-induced terpenoid responses, we analyzed traumatic oleoresin mixtures, emissions of terpenoid volatiles, and expression of terpenoid synthase (TPS) genes in Sitka spruce (Picea sitchensis) following attack by white pine weevils (Pissodes strobi) or application of MeJA. Both insects and MeJA caused traumatic resin accumulation in stems, with more accumulation induced by the weevils. Weevil-induced terpenoid emission profiles were also more complex than emissions induced by MeJA. Weevil feeding caused a rapid release of a blend of monoterpene olefins, presumably by passive evaporation of resin compounds from stem feeding sites. These compounds were not found in MeJA-induced emissions. Both weevils and MeJA caused delayed, diurnal emissions of (−)-linalool, indicating induced de novo biosynthesis of this compound. TPS transcripts strongly increased in stems upon insect attack or MeJA treatment. Time courses and intensity of induced TPS transcripts were different for monoterpene synthases, sesquiterpene synthases, and diterpene synthases. Increased levels of weevil- and MeJA-induced TPS transcripts accompanied major changes in terpenoid accumulation in stems. Induced TPS expression profiles in needles were less complex than those in stems and matched induced de novo emissions of (−)-linalool. Overall, weevils and MeJA induced similar, but not identical, terpenoid defense responses in Sitka spruce. Findings of insect- and MeJA-induced accumulation of allene oxide synthase-like and allene oxide cyclase-like transcripts are discussed in the context of traumatic resinosis and induced volatile emissions in this gymnosperm system. PMID:15618433

Functional reconstitution of cellulose synthase in Escherichia coli.

PubMed

Imai, Tomoya; Sun, Shi-Jing; Horikawa, Yoshiki; Wada, Masahisa; Sugiyama, Junji

2014-11-10

Cellulose is a high molecular weight polysaccharide of β1 → 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials.

Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria.

PubMed

Ross, Avena C; Xu, Ying; Lu, Liang; Kersten, Roland D; Shao, Zongze; Al-Suwailem, Abdulaziz M; Dorrestein, Pieter C; Qian, Pei-Yuan; Moore, Bradley S

2013-01-23

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus.

Biosynthetic Multitasking Facilitates Thalassospiramide Structural Diversity in Marine Bacteria

PubMed Central

Ross, Avena C.; Xu, Ying; Lu, Liang; Kersten, Roland D.; Shao, Zongze; Al-Suwailem, Abdulaziz M.; Dorrestein, Pieter C.; Qian, Pei-Yuan; Moore, Bradley S.

2013-01-01

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multi-module skipping and iteration. Preliminary biochemical analysis of the N-terminal NRPS module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N-terminus. PMID:23270364

Penicillospirone from a marine isolate of Penicillium sp. (SF-5292) with anti-inflammatory activity.

PubMed

Lee, Seungjun; Kim, Dong-Cheol; Park, Jin-Soo; Son, Jae-Young; Hak Sohn, Jae; Liu, Ling; Che, Yongsheng; Oh, Hyuncheol

2017-08-01

Chemical investigation of the EtOAc extract of a marine-derived fungal isolate Penicillium sp. SF-5292 yielded a new polyketide-type metabolite, penicillospirone (1). The structure of 1 was determined by analysis of spectroscopic data such as 1D and 2D NMR spectra and MS data, and the final structure including absolute configuration was unambiguously established by single-crystal X-ray diffraction analysis. In the evaluation of its anti-inflammatory effects, 1 inhibited the overproduction of nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and BV2 microglia, and these inhibitory effects were correlated with the suppressive effect of 1 against overexpressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, 1 also inhibited the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-12. Overall, the anti-inflammatory effect of 1 was suggested to be mediated through the negative regulation of NF-κB pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis

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

Yeh, Hsu-Hua; Chiang, Yi Ming; Entwistle, Ruth

2012-04-10

Genome sequencing of Aspergillus species including A. nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites have not yet been elucidated. The A. nidulans genome contains twelve nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), and fourteen NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of themore » NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in A. niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.« less

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.

Enhancing the Stress Tolerance and Virulence of an Entomopathogen by Metabolic Engineering of Dihydroxynaphthalene Melanin Biosynthesis Genes ▿ †

PubMed Central

Tseng, Min N.; Chung, Pei C.; Tzean, Shean S.

2011-01-01

Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as UV radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN) melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase genes) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin expression in the transformant of M. anisopliae was verified by spectrophotometric methods, liquid chromatography/mass spectrometry (LC/MS), and confocal microscopy. The transformant, especially under stresses, showed notably enhanced antistress capacity and virulence, in terms of germination and survival rate, infectivity, and reduced median time to death (LT50) in killing diamondback moth (Plutella xylostella) larvae compared with the wild type. The possible mechanisms in enhancing the stress tolerance and virulence, and the significance and potential for engineering melanin biosynthesis genes in other biocontrol agents and crops to improve antistress fitness are discussed. PMID:21571888

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...

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.

Characterization of bafilomycin biosynthesis in Kitasatospora setae KM-6054 and comparative analysis of gene clusters in Actinomycetales microorganisms.

PubMed

Nara, Ayako; Hashimoto, Takuya; Komatsu, Mamoru; Nishiyama, Makoto; Kuzuyama, Tomohisa; Ikeda, Haruo

2017-05-01

Bafilomycins A 1 , C 1 and B 1 (setamycin) produced by Kitasatospora setae KM-6054 belong to the plecomacrolide family, which exhibit antibacterial, antifungal, antineoplastic and immunosuppressive activities. An analysis of gene clusters from K. setae KM-6054 governing the biosynthesis of bafilomycins revealed that it contains five large open reading frames (ORFs) encoding the multifunctional polypeptides of bafilomycin polyketide synthases (PKSs). These clustered PKS genes, which are responsible for bafilomycin biosynthesis, together encode 11 homologous sets of enzyme activities, each catalyzing a specific round of polyketide chain elongation. The region contains an additional 13 ORFs spanning a distance of 73 287 bp, some of which encode polypeptides governing other key steps in bafilomycin biosynthesis. Five ORFs, BfmB, BfmC, BfmD, BfmE and BfmF, were involved in the formation of methoxymalonyl-acyl carrier protein (ACP). Two possible regulatory genes, bfmR and bfmH, were found downstream of the above genes. A gene-knockout analysis revealed that BfmR was only a transcriptional regulator for the transcription of bafilomycin biosynthetic genes. Two genes, bfmI and bfmJ, were found downstream of bfmH. An analysis of these gene-disruption mutants in addition to an enzymatic analysis of BfmI and BfmJ revealed that BfmJ activated fumarate and BfmI functioned as a catalyst to form a fumaryl ester at the C21 hydroxyl residue of bafilomycin A 1 . A comparative analysis of bafilomycin gene clusters in K. setae KM-6054, Streptomyces lohii JCM 14114 and Streptomyces griseus DSM 2608 revealed that each ORF of both gene clusters in two Streptomyces strains were quite similar to each other. However, each ORF of gene cluster in K. setae KM-6054 was of lower similarity to that of corresponding ORF in the two Streptomyces species.

Antibacterial and Antioxidant Activities of Novel Actinobacteria Strain Isolated from Gulf of Khambhat, Gujarat.

PubMed

Dholakiya, Riddhi N; Kumar, Raghawendra; Mishra, Avinash; Mody, Kalpana H; Jha, Bhavanath

2017-01-01

Bacterial secondary metabolites possess a wide range of biologically active compounds including antibacterial and antioxidants. In this study, a Gram-positive novel marine Actinobacteria was isolated from sea sediment which showed 84% 16S rRNA gene sequence (KT588655) similarity with Streptomyces variabilis (EU841661) and designated as Streptomyces variabilis RD-5. The genus Streptomyces is considered as a promising source of bioactive secondary metabolites. The isolated novel bacterial strain was characterized by antibacterial characteristics and antioxidant activities. The BIOLOG based analysis suggested that S. variabilis RD-5 utilized a wide range of substrates compared to the reference strain. The result is further supported by statistical analysis such as AWCD (average well color development), heat-map and PCA (principal component analysis). The whole cell fatty acid profiling showed the dominance of iso/anteiso branched C15-C17 long chain fatty acids. The identified strain S. variabilis RD-5 exhibited a broad spectrum of antibacterial activities for the Gram-negative bacteria ( Escherichia coli NCIM 2065, Shigella boydii NCIM, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas sp. NCIM 2200 and Salmonella enteritidis NCIM), and Gram-positive bacteria ( Bacillus subtilis NCIM 2920 and Staphylococcus aureus MTCC 96). Extract of S. variabilis strain RD-5 showed 82.86 and 89% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and metal chelating activity, respectively, at 5.0 mg/mL. While H 2 O 2 scavenging activity was 74.5% at 0.05 mg/mL concentration. Furthermore, polyketide synthases (PKSs types I and II), an enzyme complex that produces polyketides, the encoding gene(s) detected in the strain RD-5 which may probably involve for the synthesis of antibacterial compound(s). In conclusion, a novel bacterial strain of Actinobacteria , isolated from the unexplored sea sediment of Alang, Gulf of Khambhat (Gujarat), India showed promising antibacterial activities. However, fractionation and further characterization of active compounds from S. variabilis RD-5 are needed for their optimum utilization toward antibacterial purposes.

Antibacterial and Antioxidant Activities of Novel Actinobacteria Strain Isolated from Gulf of Khambhat, Gujarat

PubMed Central

Dholakiya, Riddhi N.; Kumar, Raghawendra; Mishra, Avinash; Mody, Kalpana H.; Jha, Bhavanath

2017-01-01

Bacterial secondary metabolites possess a wide range of biologically active compounds including antibacterial and antioxidants. In this study, a Gram-positive novel marine Actinobacteria was isolated from sea sediment which showed 84% 16S rRNA gene sequence (KT588655) similarity with Streptomyces variabilis (EU841661) and designated as Streptomyces variabilis RD-5. The genus Streptomyces is considered as a promising source of bioactive secondary metabolites. The isolated novel bacterial strain was characterized by antibacterial characteristics and antioxidant activities. The BIOLOG based analysis suggested that S. variabilis RD-5 utilized a wide range of substrates compared to the reference strain. The result is further supported by statistical analysis such as AWCD (average well color development), heat-map and PCA (principal component analysis). The whole cell fatty acid profiling showed the dominance of iso/anteiso branched C15–C17 long chain fatty acids. The identified strain S. variabilis RD-5 exhibited a broad spectrum of antibacterial activities for the Gram-negative bacteria (Escherichia coli NCIM 2065, Shigella boydii NCIM, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas sp. NCIM 2200 and Salmonella enteritidis NCIM), and Gram-positive bacteria (Bacillus subtilis NCIM 2920 and Staphylococcus aureus MTCC 96). Extract of S. variabilis strain RD-5 showed 82.86 and 89% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and metal chelating activity, respectively, at 5.0 mg/mL. While H2O2 scavenging activity was 74.5% at 0.05 mg/mL concentration. Furthermore, polyketide synthases (PKSs types I and II), an enzyme complex that produces polyketides, the encoding gene(s) detected in the strain RD-5 which may probably involve for the synthesis of antibacterial compound(s). In conclusion, a novel bacterial strain of Actinobacteria, isolated from the unexplored sea sediment of Alang, Gulf of Khambhat (Gujarat), India showed promising antibacterial activities. However, fractionation and further characterization of active compounds from S. variabilis RD-5 are needed for their optimum utilization toward antibacterial purposes. PMID:29270160

Pseudomonas pseudoalcaligenes CECT5344, a cyanide-degrading bacterium with by-product (polyhydroxyalkanoates) formation capacity.

PubMed

Manso Cobos, Isabel; Ibáñez García, María Isabel; de la Peña Moreno, Fernando; Sáez Melero, Lara Paloma; Luque-Almagro, Víctor Manuel; Castillo Rodríguez, Francisco; Roldán Ruiz, María Dolores; Prieto Jiménez, María Auxiliadora; Moreno Vivián, Conrado

2015-06-10

Cyanide is one of the most toxic chemicals produced by anthropogenic activities like mining and jewelry industries, which generate wastewater residues with high concentrations of this compound. Pseudomonas pseudoalcaligenes CECT5344 is a model microorganism to be used in detoxification of industrial wastewaters containing not only free cyanide (CN(-)) but also cyano-derivatives, such as cyanate, nitriles and metal-cyanide complexes. Previous in silico analyses suggested the existence of genes putatively involved in metabolism of short chain length (scl-) and medium chain length (mcl-) polyhydroxyalkanoates (PHAs) located in three different clusters in the genome of this bacterium. PHAs are polyesters considered as an alternative of petroleum-based plastics. Strategies to optimize the bioremediation process in terms of reducing the cost of the production medium are required. In this work, a biological treatment of the jewelry industry cyanide-rich wastewater coupled to PHAs production as by-product has been considered. The functionality of the pha genes from P. pseudoalcaligenes CECT5344 has been demonstrated. Mutant strains defective in each proposed PHA synthases coding genes (Mpha(-), deleted in putative mcl-PHA synthases; Spha(-), deleted in the putative scl-PHA synthase) were generated. The accumulation and monomer composition of scl- or mcl-PHAs in wild type and mutant strains were confirmed by gas chromatography-mass spectrometry (GC-MS). The production of PHAs as by-product while degrading cyanide from the jewelry industry wastewater was analyzed in batch reactor in each strain. The wild type and the mutant strains grew at similar rates when using octanoate as the carbon source and cyanide as the sole nitrogen source. When cyanide was depleted from the medium, both scl-PHAs and mcl-PHAs were detected in the wild-type strain, whereas scl-PHAs or mcl-PHAs were accumulated in Mpha(-) and Spha(-), respectively. The scl-PHAs were identified as homopolymers of 3-hydroxybutyrate and the mcl-PHAs were composed of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers. These results demonstrated, as proof of concept, that talented strains such as P. pseudoalcaligenes might be applied in bioremediation of industrial residues containing cyanide, while concomitantly generate by-products like polyhydroxyalkanoates. A customized optimization of the target bioremediation process is required to gain benefits of this type of approaches.

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.

Detoxification of polycyclic aromatic hydrocarbons (PAHs) in Arabidopsis thaliana involves a putative flavonol synthase.

PubMed

Hernández-Vega, Juan C; Cady, Brian; Kayanja, Gilbert; Mauriello, Anthony; Cervantes, Natalie; Gillespie, Andrea; Lavia, Lisa; Trujillo, Joshua; Alkio, Merianne; Colón-Carmona, Adán

2017-01-05

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants with cytotoxic, teratogenic and carcinogenic properties. Bioremediation studies with bacteria have led to the identification of dioxygenases (DOXs) in the first step to degrade these recalcitrant compounds. In this study, we characterized the role of the Arabidopsis thaliana AT5G05600, a putative DOX of the flavonol synthase family, in the transformation of PAHs. Phenotypic analysis of loss-of-function mutant lines showed that these plant lines were less sensitive to the toxic effects of phenanthrene, suggesting possible roles of this gene in PAH degradation in vivo. Interestingly, these mutant lines showed less accumulation of H 2 O 2 after PAH exposure. Transgenic lines over-expressing At5g05600 showed a hypersensitive response and more oxidative stress after phenanthrene treatments. Moreover, fluorescence spectra results of biochemical assays with the recombinant His-tagged protein AT5G05600 detected chemical modifications of phenanthrene. Taken together, these results support the hypothesis that AT5G05600 is involved in the catabolism of PAHs and the accumulation of toxic intermediates during PAH biotransformation in plants. This research represents the first step in the design of transgenic plants with the potential to degrade PAHs, leading to the development of vigorous plant varieties that can reduce the levels of these pollutants in the environment. Copyright © 2016 Elsevier B.V. All rights reserved.

Crystallization and X-ray diffraction analysis of a putative bacterial class I labdane-related diterpene synthase.

PubMed

Serrano-Posada, Hugo; Centeno-Leija, Sara; Rojas-Trejo, Sonia; Stojanoff, Vivian; Rodríguez-Sanoja, Romina; Rudiño-Piñera, Enrique; Sánchez, Sergio

2015-09-01

Labdane-related diterpenoids are natural products with potential pharmaceutical applications that are rarely found in bacteria. Here, a putative class I labdane-related diterpene synthase (LrdC) identified by genome mining in a streptomycete was successfully crystallized using the microbatch method. Crystals of the LrdC enzyme were obtained in a holo form with its natural cofactor Mg(2+) (LrdC-Mg(2+)) and in complex with inorganic pyrophosphate (PPi) (LrdC-Mg(2+)-PPi). Crystals of native LrdC-Mg(2+) diffracted to 2.50 Å resolution and belonged to the trigonal space group P3221, with unit-cell parameters a = b = 107.1, c = 89.2 Å. Crystals of the LrdC-Mg(2+)-PPi complex grown in the same conditions as the native enzyme with PEG 8000 diffracted to 2.36 Å resolution and also belonged to the trigonal space group P3221. Crystals of the LrdC-Mg(2+)-PPi complex grown in a second crystallization condition with PEG 3350 diffracted to 2.57 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 49.9, b = 104.1, c = 66.5 Å, β = 111.4°. The structure was determined by the single-wavelength anomalous dispersion (SAD) technique using the osmium signal from a potassium hexachloroosmate (IV) derivative.

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.

Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

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

Rice, E.A.; Bannon, G.A.; Glenn, K.C.

2008-11-21

The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysinemore » revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.« less

Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis.

PubMed

Yamamura, Yoshimi; Kurosaki, Fumiya; Lee, Jung-Bum

2017-03-07

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis.

Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis

PubMed Central

Yamamura, Yoshimi; Kurosaki, Fumiya; Lee, Jung-Bum

2017-01-01

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis. PMID:28266568

New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145.

PubMed

Kato, Hiroki; Tsunematsu, Yuta; Yamamoto, Tsuyoshi; Namiki, Takuya; Kishimoto, Shinji; Noguchi, Hiroshi; Watanabe, Kenji

2016-07-01

To rapidly identify novel natural products and their associated biosynthetic genes from underutilized and genetically difficult-to-manipulate microbes, we developed a method that uses (1) chemical screening to isolate novel microbial secondary metabolites, (2) bioinformatic analyses to identify a potential biosynthetic gene cluster and (3) heterologous expression of the genes in a convenient host to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. The chemical screen was achieved by searching known natural product databases with data from liquid chromatographic and high-resolution mass spectrometric analyses collected on the extract from a target microbe culture. Using this method, we were able to isolate two new meroterpenes, subglutinols C (1) and D (2), from an entomopathogenic filamentous fungus Metarhizium robertsii ARSEF 23. Bioinformatics analysis of the genome allowed us to identify a gene cluster likely to be responsible for the formation of subglutinols. Heterologous expression of three genes from the gene cluster encoding a polyketide synthase, a prenyltransferase and a geranylgeranyl pyrophosphate synthase in Aspergillus nidulans A1145 afforded an α-pyrone-fused uncyclized diterpene, the expected intermediate of the subglutinol biosynthesis, thereby confirming the gene cluster to be responsible for the subglutinol biosynthesis. These results indicate the usefulness of our methodology in isolating new natural products and identifying their associated biosynthetic gene cluster from microbes that are not amenable to genetic manipulation. Our method should facilitate the natural product discovery efforts by expediting the identification of new secondary metabolites and their associated biosynthetic genes from a wider source of microbes.

A new strategy for strain improvement of Aurantiochytrium sp. based on heavy-ions mutagenesis and synergistic effects of cold stress and inhibitors of enoyl-ACP reductase.

PubMed

Cheng, Yu-Rong; Sun, Zhi-Jie; Cui, Gu-Zhen; Song, Xiaojin; Cui, Qiu

2016-11-01

Developing a strain with high docosahexaenoic acid (DHA) yield and stable fermenting-performance is an imperative way to improve DHA production using Aurantiochytrium sp., a microorganism with two fatty acid synthesis pathways: polyketide synthase (PKS) pathway and Type I fatty acid synthase (FAS) pathway. This study investigated the growth and metabolism response of Aurantiochytrium sp. CGMCC 6208 to two inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan), and proposed a method of screening high DHA yield Aurantiochytrium sp. strains with heavy ion mutagenesis and pre-selection by synergistic usage of cold stress (4°C) and FAS inhibitors (triclosan and isoniazid). Results showed that (1) isoniazid and triclosan have positive effects on improving DHA level of cells; (2) mutants from irradiation dosage of 120Gy yielded more DHA compared with cells from 40Gy, 80Gy treatment and wild type; (3) DHA contents of mutants pre-selected by inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan)at 4°C, were significantly higher than that of wild type; (4) compared to the wild type, the DHA productivity and yield of a mutant (T-99) obtained from Aurantiochytrium sp. CGMCC 6208 by the proposed method increased by 50% from 0.18 to 0.27g/Lh and 30% from 21 to 27g/L, respectively. In conclusion, this study developed a feasible method to screen Aurantiochytrium sp. with high DHA yield by a combination of heavy-ion mutagenesis and mutant-preselection by FAS inhibitors and cold stress. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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.

Ethanol Dose- and Time-dependently Increases α and β Subunits of Mitochondrial ATP Synthase of Cultured Neonatal Rat Cardiomyocytes.

PubMed

Mashimo, Keiko; Arthur, Peter G; Ohno, Youkichi

2015-01-01

Mitochondria are target subcellular organelles of ethanol. In this study, the effects of ethanol on protein composition was examined with 2-dimensional electrophoresis of protein extracts from cultured neonatal rat cardiomyocytes exposed to 100 mM ethanol for 24 hours. A putative β subunit of mitochondrial ATP synthase was increased, which was confirmed by Western blot. The cellular protein abundances in the α and β subunits of ATP synthase increased in dose (0, 10, 50, and 100 mM) - and time (0.5 hour and 24 hours) -dependent manners. The DNA microarray analysis of total RNA extract demonstrated that gene expression of the corresponding messenger RNAs of these subunit proteins did not significantly alter due to 24-hour ethanol exposure. Therefore, protein expression of these nuclear-encoded mitochondrial proteins may be regulated at the translational, rather than the transcriptional, level. Alternatively, degradation of these subunit proteins might be decreased. Additionally, cellular ATP content of cardiomyocytes scarcely decreased following 24-hour exposure to any examined concentrations of ethanol. Previous studies, together with this study, have demonstrated that protein abundance of the α subunit or β subunit or both subunits of ATP synthase after ethanol exposure or dysfunctional conditions might differ according to tissue: significant increases in heart but decreases in liver and brain. Thus, it is suggested that the abundance of subunit proteins of mitochondrial ATP synthase in the ethanol-exposed heart, being different from that in the liver and brain, should increase dose-dependently through either translational upregulation or decreased degradation or both to maintain ATP production, as the heart requires much more energy than other tissues for continuing sustained contractions.

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

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

Against All Odds: Trehalose-6-Phosphate Synthase and Trehalase Genes in the Bdelloid Rotifer Adineta vaga Were Acquired by Horizontal Gene Transfer and Are Upregulated during Desiccation

PubMed Central

Hespeels, Boris; Li, Xiang; Flot, Jean-François; Pigneur, Lise-Marie; Malaisse, Jeremy; Da Silva, Corinne; Van Doninck, Karine

2015-01-01

The disaccharide sugar trehalose is essential for desiccation resistance in most metazoans that survive dryness; however, neither trehalose nor the enzymes involved in its metabolism have ever been detected in bdelloid rotifers despite their extreme resistance to desiccation. Here we screened the genome of the bdelloid rotifer Adineta vaga for genes involved in trehalose metabolism. We discovered a total of four putative trehalose-6-phosphate synthase (TPS) and seven putative trehalase (TRE) gene copies in the genome of this ameiotic organism; however, no trehalose-6-phosphate phosphatase (TPP) gene or domain was detected. The four TPS copies of A. vaga appear more closely related to plant and fungi proteins, as well as to some protists, whereas the seven TRE copies fall in bacterial clades. Therefore, A. vaga likely acquired its trehalose biosynthesis and hydrolysis genes by horizontal gene transfers. Nearly all residues important for substrate binding in the predicted TPS domains are highly conserved, supporting the hypothesis that several copies of the genes might be functional. Besides, RNAseq library screening showed that trehalase genes were highly expressed compared to TPS genes, explaining probably why trehalose had not been detected in previous studies of bdelloids. A strong overexpression of their TPS genes was observed when bdelloids enter desiccation, suggesting a possible signaling role of trehalose-6-phosphate or trehalose in this process. PMID:26161530

The genetic basis of natural variation for iron homeostasis in the maize IBM population

PubMed Central

2014-01-01

Background Iron (Fe) deficiency symptoms in maize (Zea mays subsp. mays) express as leaf chlorosis, growth retardation, as well as yield reduction and are typically observed when plants grow in calcareous soils at alkaline pH. To improve our understanding of genotypical variability in the tolerance to Fe deficiency-induced chlorosis, the objectives of this study were to (i) determine the natural genetic variation of traits related to Fe homeostasis in the maize intermated B73 × Mo17 (IBM) population, (ii) to identify quantitative trait loci (QTLs) for these traits, and (iii) to analyze expression levels of genes known to be involved in Fe homeostasis as well as of candidate genes obtained from the QTL analysis. Results In hydroponically-grown maize, a total of 47 and 39 QTLs were detected for the traits recorded under limited and adequate supply of Fe, respectively. Conclusions From the QTL results, we were able to identify new putative candidate genes involved in Fe homeostasis under a deficient or adequate Fe nutritional status, like Ferredoxin class gene, putative ferredoxin PETF, metal tolerance protein MTP4, and MTP8. Furthermore, our expression analysis of candidate genes suggested the importance of trans-acting regulation for 2’-deoxymugineic acid synthase 1 (DMAS1), nicotianamine synthase (NAS3, NAS1), formate dehydrogenase 1 (FDH1), methylthioribose-1-phosphate isomerase (IDI2), aspartate/tyrosine/aromatic aminotransferase (IDI4), and methylthioribose kinase (MTK). PMID:24400634

The Eucalyptus terpene synthase gene family.

PubMed

Külheim, Carsten; Padovan, Amanda; Hefer, Charles; Krause, Sandra T; Köllner, Tobias G; Myburg, Alexander A; Degenhardt, Jörg; Foley, William J

2015-06-11

Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts. The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus. Our data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.

The First Step of Gibberellin Biosynthesis in Pumpkin Is Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by Differentially Regulated Genes

PubMed Central

Smith, Maria W.; Yamaguchi, Shinjiro; Ait-Ali, Tahar; Kamiya, Yuji

1998-01-01

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression. PMID:9847116

Rewiring a secondary metabolite pathway towards itaconic acid production in Aspergillus niger.

PubMed

Hossain, Abeer H; Li, An; Brickwedde, Anja; Wilms, Lars; Caspers, Martien; Overkamp, Karin; Punt, Peter J

2016-07-28

The industrially relevant filamentous fungus Aspergillus niger is widely used in industry for its secretion capabilities of enzymes and organic acids. Biotechnologically produced organic acids promise to be an attractive alternative for the chemical industry to replace petrochemicals. Itaconic acid (IA) has been identified as one of the top twelve building block chemicals which have high potential to be produced by biotechnological means. The IA biosynthesis cluster (cadA, mttA and mfsA) has been elucidated in its natural producer Aspergillus terreus and transferred to A. niger to enable IA production. Here we report the rewiring of a secondary metabolite pathway towards further improved IA production through the overexpression of a putative cytosolic citrate synthase citB in a A. niger strain carrying the IA biosynthesis cluster. We have previously shown that expression of cadA from A. terreus results in itaconic acid production in A. niger AB1.13, albeit at low levels. This low-level production is boosted fivefold by the overexpression of mttA and mfsA in itaconic acid producing AB1.13 CAD background strains. Controlled batch cultivations with AB1.13 CAD + MFS + MTT strains showed increased production of itaconic acid compared with AB1.13 CAD strain. Moreover, preliminary RNA-Seq analysis of an itaconic acid producing AB1.13 CAD strain has led to the identification of the putative cytosolic citrate synthase citB which was induced in an IA producing strain. We have overexpressed citB in a AB1.13 CAD + MFS + MTT strain and by doing so hypothesize to have targeted itaconic acid production to the cytosolic compartment. By overexpressing citB in AB1.13 CAD + MFS + MTT strains in controlled batch cultivations we have achieved highly increased titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h while no CA was produced. Expression of the IA biosynthesis cluster in Aspergillus niger AB1.13 strain enables IA production. Moreover, in the AB1.13 CAD strain IA production resulted in overexpression of a putative cytosolic citrate synthase citB. Upon overexpression of citB we have achieved titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h in controlled batch cultivations. By overexpressing citB we have also diminished side product formation and optimized the production pathway towards IA.

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.

Modeling linear and cyclic PKS intermediates through atom replacement.

PubMed

Shakya, Gaurav; Rivera, Heriberto; Lee, D John; Jaremko, Matt J; La Clair, James J; Fox, Daniel T; Haushalter, Robert W; Schaub, Andrew J; Bruegger, Joel; Barajas, Jesus F; White, Alexander R; Kaur, Parminder; Gwozdziowski, Emily R; Wong, Fiona; Tsai, Shiou-Chuan; Burkart, Michael D

2014-12-03

The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs.

Roles of type II thioesterases and their application for secondary metabolite yield improvement.

PubMed

Kotowska, Magdalena; Pawlik, Krzysztof

2014-09-01

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

Phylogenetic Studies, Gene Cluster Analysis, and Enzymatic Reaction Support Anthrahydroquinone Reduction as the Physiological Function of Fungal 17β-Hydroxysteroid Dehydrogenase.

PubMed

Fürtges, Leon; Conradt, David; Schätzle, Michael A; Singh, Shailesh Kumar; Kraševec, Nada; Rižner, Tea Lanišnik; Müller, Michael; Husain, Syed Masood

2017-01-03

17β-Hydroxysteroid dehydrogenase (17β-HSDcl) from the filamentous fungus Curvularia lunata (teleomorph Cochliobolus lunatus) catalyzes NADP(H)-dependent oxidoreductions of androgens and estrogens. Despite detailed biochemical and structural characterization of 17β-HSDcl, its physiological function remains unknown. On the basis of amino acid sequence alignment, phylogenetic studies, and the recent identification of the physiological substrates of the homologous MdpC from Aspergillus nidulans and AflM from Aspergillus parasiticus, we propose an anthrahydroquinone as the physiological substrate of 17β-HSDcl. This is also supported by our analysis of a secondary metabolite biosynthetic gene cluster in C. lunata m118, containing 17β-HSDcl and ten other genes, including a polyketide synthase probably involved in emodin formation. Chemoenzymatic reduction of emodin by 17β-HSDcl in the presence of sodium dithionite verified this hypothesis. On the basis of these results, the involvement of a 17β-HSDcl in the biosynthesis of other anthrahydroquinone-derived natural products is proposed; hence, 17β-HSDcl should be more appropriately referred to as a polyhydroxyanthracene reductase (PHAR). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The modules of trans-acyltransferase assembly lines redefined with a central acyl carrier protein.

PubMed

Vander Wood, Drew A; Keatinge-Clay, Adrian T

2018-06-01

Here, the term "module" is redefined for trans-acyltransferase (trans-AT) assembly lines to agree with how its domains cooperate and evolutionarily co-migrate. The key domain in both the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) modules of assembly lines is the acyl carrier protein (ACP). ACPs not only relay growing acyl chains through the assembly line but also collaborate with enzymes in modules, both in cis and in trans, to add a specific chemical moiety. A ketosynthase (KS) downstream of ACP often plays the role of gatekeeper, ensuring that only a single intermediate generated by the enzymes of a module is passed downstream. Bioinformatic analysis of 526 ACPs from 33 characterized trans-AT assembly lines reveals ACPs from the same module type generally clade together, reflective of the co-evolution of these domains with their cognate enzymes. While KSs downstream of ACPs from the same module type generally also clade together, KSs upstream of ACPs do not-in disagreement with the traditional definition of a module. Beyond nomenclature, the presented analysis impacts our understanding of module function, the evolution of assembly lines, pathway prediction, and assembly line engineering. © 2018 Wiley Periodicals, Inc.

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

Investigation of the Biosynthetic Potential of Endophytes in Traditional Chinese Anticancer Herbs

PubMed Central

Miller, Kristin I.; Qing, Chen; Sze, Daniel Man Yuen; Neilan, Brett A.

2012-01-01

Traditional Chinese medicine encompasses a rich empirical knowledge of the use of plants for the treatment of disease. In addition, the microorganisms associated with medicinal plants are also of interest as the producers of the compounds responsible for the observed plant bioactivity. The present study has pioneered the use of genetic screening to assess the potential of endophytes to synthesize bioactive compounds, as indicated by the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes. The total DNA extracts of 30 traditional Chinese herbs, were screened for functional genes involved in the biosynthesis of bioactive compounds. The four PCR screens were successful in targeting four bacterial PKS, six bacterial NRPS, ten fungal PKS and three fungal NRPS gene fragments. Analysis of the detected endophyte gene fragments afforded consideration of the possible bioactivity of the natural products produced by endophytes in medicinal herbs. This investigation describes a rapid method for the initial screening of medicinal herbs and has highlighted a subset of those plants that host endophytes with biosynthetic potential. These selected plants can be the focus of more comprehensive endophyte isolation and natural product studies. PMID:22629306

The Kolumbo submarine volcano of Santorini island is a large pool of bacterial strains with antimicrobial activity.

PubMed

Bourbouli, Maria; Katsifas, Efstathios A; Papathanassiou, Evangelos; Karagouni, Amalia D

2015-05-01

Microbes in hydrothermal vents with their unique secondary metabolism may represent an untapped potential source of new natural products. In this study, samples were collected from the hydrothermal field of Kolumbo submarine volcano in the Aegean Sea, in order to isolate bacteria with antimicrobial activity. Eight hundred and thirty-two aerobic heterotrophic bacteria were isolated and then differentiated through BOX-PCR analysis at the strain level into 230 genomic fingerprints, which were screened against 13 different type strains (pathogenic and nonpathogenic) of Gram-positive, Gram-negative bacteria and fungi. Forty-two out of 176 bioactive-producing genotypes (76 %) exhibited antimicrobial activity against at least four different type strains and were selected for 16S rDNA sequencing and screening for nonribosomal peptide (NRPS) and polyketide (PKS) synthases genes. The isolates were assigned to genus Bacillus and Proteobacteria, and 20 strains harbored either NRPS, PKS type I or both genes. This is the first report on the diversity of culturable mesophilic bacteria associated with antimicrobial activity from Kolumbo area; the extremely high proportion of antimicrobial-producing strains suggested that this unique environment may represent a potential reservoir of novel bioactive compounds.

Development of a one-step gene knock-out and knock-in method for metabolic engineering of Aureobasidium pullulans.

PubMed

Guo, Jian; Wang, Yuanhua; Li, Baozhong; Huang, Siyao; Chen, Yefu; Guo, Xuewu; Xiao, Dongguang

2017-06-10

Aureobasidium pullulans is an increasingly attractive host for bio-production of pullulan, heavy oil, polymalic acid, and a large spectrum of extracellular enzymes. To date, genetic manipulation of A. pullulans mainly relies on time-consuming conventional restriction enzyme digestion and ligation methods. In this study, we present a one-step homologous recombination-based method for rapid genetic manipulation in A. pullulans. Overlaps measuring >40bp length and 10μg DNA segments for homologous recombination provided maximum benefits to transformation of A. pullulans. This optimized method was successfully applied to PKSIII gene (encodes polyketide synthase) knock-out and gltP gene (encodes glycolipid transfer protein) knock-in. After disruption of PKSIII gene, secretion of melanin decreased slightly. The melanin purified from disruptant showed lower reducing capacity compared with that of the parent strain, leading to a decrease in exopolysaccharide production. Knock-in of gltP gene resulted in at least 4.68-fold increase in heavy oil production depending on the carbon source used, indicating that gltP can regulate heavy oil synthesis in A. pullulans. Copyright © 2017 Elsevier B.V. All rights reserved.

Littoral lichens as a novel source of potentially bioactive Actinobacteria.

PubMed

Parrot, Delphine; Antony-Babu, Sanjay; Intertaglia, Laurent; Grube, Martin; Tomasi, Sophie; Suzuki, Marcelino T

2015-10-30

Cultivable Actinobacteria are the largest source of microbially derived bioactive molecules. The high demand for novel antibiotics highlights the need for exploring novel sources of these bacteria. Microbial symbioses with sessile macro-organisms, known to contain bioactive compounds likely of bacterial origin, represent an interesting and underexplored source of Actinobacteria. We studied the diversity and potential for bioactive-metabolite production of Actinobacteria associated with two marine lichens (Lichina confinis and L. pygmaea; from intertidal and subtidal zones) and one littoral lichen (Roccella fuciformis; from supratidal zone) from the Brittany coast (France), as well as the terrestrial lichen Collema auriforme (from a riparian zone, Austria). A total of 247 bacterial strains were isolated using two selective media. Isolates were identified and clustered into 101 OTUs (98% identity) including 51 actinobacterial OTUs. The actinobacterial families observed were: Brevibacteriaceae, Cellulomonadaceae, Gordoniaceae, Micrococcaceae, Mycobacteriaceae, Nocardioidaceae, Promicromonosporaceae, Pseudonocardiaceae, Sanguibacteraceae and Streptomycetaceae. Interestingly, the diversity was most influenced by the selective media rather than lichen species or the level of lichen thallus association. The potential for bioactive-metabolite biosynthesis of the isolates was confirmed by screening genes coding for polyketide synthases types I and II. These results show that littoral lichens are a source of diverse potentially bioactive Actinobacteria.

Littoral lichens as a novel source of potentially bioactive Actinobacteria

PubMed Central

Parrot, Delphine; Antony-Babu, Sanjay; Intertaglia, Laurent; Grube, Martin; Tomasi, Sophie; Suzuki, Marcelino T.

2015-01-01

Cultivable Actinobacteria are the largest source of microbially derived bioactive molecules. The high demand for novel antibiotics highlights the need for exploring novel sources of these bacteria. Microbial symbioses with sessile macro-organisms, known to contain bioactive compounds likely of bacterial origin, represent an interesting and underexplored source of Actinobacteria. We studied the diversity and potential for bioactive-metabolite production of Actinobacteria associated with two marine lichens (Lichina confinis and L. pygmaea; from intertidal and subtidal zones) and one littoral lichen (Roccella fuciformis; from supratidal zone) from the Brittany coast (France), as well as the terrestrial lichen Collema auriforme (from a riparian zone, Austria). A total of 247 bacterial strains were isolated using two selective media. Isolates were identified and clustered into 101 OTUs (98% identity) including 51 actinobacterial OTUs. The actinobacterial families observed were: Brevibacteriaceae, Cellulomonadaceae, Gordoniaceae, Micrococcaceae, Mycobacteriaceae, Nocardioidaceae, Promicromonosporaceae, Pseudonocardiaceae, Sanguibacteraceae and Streptomycetaceae. Interestingly, the diversity was most influenced by the selective media rather than lichen species or the level of lichen thallus association. The potential for bioactive-metabolite biosynthesis of the isolates was confirmed by screening genes coding for polyketide synthases types I and II. These results show that littoral lichens are a source of diverse potentially bioactive Actinobacteria. PMID:26514347

Endophytic Paraconiothyrium sp. from Zingiber officinale Rosc. Displays Broad-Spectrum Antimicrobial Activity by Production of Danthron.

PubMed

Anisha, C; Sachidanandan, P; Radhakrishnan, E K

2018-03-01

The bioactivity spectrum of fungal endophytes isolated from Zingiber officinale was analyzed against clinical pathogens and against the phytopathogen Pythium myriotylum, which causes Pythium rot in ginger. One of the isolates GFM13 showed broad bioactivity against various pathogens tested including P. myriotylum. The spore suspension as well as the culture filtrate of the endophytic fungal isolate was found to effectively protect ginger rhizomes from Pythium rot. By molecular identification, the fungal endophyte was identified as Paraconiothyrium sp. The bioactive compound produced by the isolate was separated by bioactivity-guided fractionation and was identified by GC-MS as danthron, an anthraquinone derivative. PCR amplification showed the presence of non-reducing polyketide synthase gene (NR-PKS) in the endophyte GFM13, which is reported to be responsible for the synthesis of anthraquinones in fungi. This is the first report of danthron being produced as the biologically active component of Paraconiothyrium sp. Danthron is reported to have wide pharmaceutical and agronomic applications which include its use as a fungicide in agriculture. The broad-spectrum antimicrobial activity of danthron and the endophytic origin of Paraconiothyrium sp. offer immense applications of the study.

Shotgun proteomic analysis of Emiliania huxleyi, a marine phytoplankton species of major biogeochemical importance.

PubMed

Jones, Bethan M; Edwards, Richard J; Skipp, Paul J; O'Connor, C David; Iglesias-Rodriguez, M Debora

2011-06-01

Emiliania huxleyi is a unicellular marine phytoplankton species known to play a significant role in global biogeochemistry. Through the dual roles of photosynthesis and production of calcium carbonate (calcification), carbon is transferred from the atmosphere to ocean sediments. Almost nothing is known about the molecular mechanisms that control calcification, a process that is tightly regulated within the cell. To initiate proteomic studies on this important and phylogenetically remote organism, we have devised efficient protein extraction protocols and developed a bioinformatics pipeline that allows the statistically robust assignment of proteins from MS/MS data using preexisting EST sequences. The bioinformatics tool, termed BUDAPEST (Bioinformatics Utility for Data Analysis of Proteomics using ESTs), is fully automated and was used to search against data generated from three strains. BUDAPEST increased the number of identifications over standard protein database searches from 37 to 99 proteins when data were amalgamated. Proteins involved in diverse cellular processes were uncovered. For example, experimental evidence was obtained for a novel type I polyketide synthase and for various photosystem components. The proteomic and bioinformatic approaches developed in this study are of wider applicability, particularly to the oceanographic community where genomic sequence data for species of interest are currently scarce.

Insights into a divergent phenazine biosynthetic pathway governed by a plasmid-born esmeraldin gene cluster.

PubMed

Rui, Zhe; Ye, Min; Wang, Shuoguo; Fujikawa, Kaori; Akerele, Bankole; Aung, May; Floss, Heinz G; Zhang, Wenjun; Yu, Tin-Wein

2012-09-21

Phenazine-type metabolites arise from either phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC). Although the biosynthesis of PCA has been studied extensively, PDC assembly remains unclear. Esmeraldins and saphenamycin, the PDC originated products, are antimicrobial and antitumor metabolites isolated from Streptomyces antibioticus Tü 2706. Herein, the esmeraldin biosynthetic gene cluster was identified on a dispensable giant plasmid. Twenty-four putative esm genes were characterized by bioinformatics, mutagenesis, genetic complementation, and functional protein expressions. Unlike enzymes involved in PCA biosynthesis, EsmA1 and EsmA2 together decisively promoted the PDC yield. The resulting PDC underwent a series of conversions to give 6-acetylphenazine-1-carboxylic acid, saphenic acid, and saphenamycin through a unique one-carbon extension by EsmB1-B5, a keto reduction by EsmC, and an esterification by EsmD1-D3, the atypical polyketide sythases, respectively. Two transcriptional regulators, EsmT1 and EsmT2, are required for esmeraldin production. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

AtCSLA7, a Cellulose Synthase-Like Putative Glycosyltransferase, Is Important for Pollen Tube Growth and Embryogenesis in Arabidopsis1

PubMed Central

Goubet, Florence; Misrahi, Audrey; Park, Soon Ki; Zhang, Zhinong; Twell, David; Dupree, Paul

2003-01-01

The cellulose synthase-like proteins are a large family of proteins in plants thought to be processive polysaccharide β-glycosyltransferases. We have characterized an Arabidopsis mutant with a transposon insertion in the gene encoding AtCSLA7 of the CSLA subfamily. Analysis of the transmission efficiency of the insertion indicated that AtCSLA7 is important for pollen tube growth. Moreover, the homozygous insertion was embryo lethal. A detailed analysis of seed developmental progression revealed that mutant embryos developed more slowly than wild-type siblings. The mutant embryos also showed abnormal cell patterning and they arrested at a globular stage. The defective embryonic development was associated with reduced proliferation and failed cellularization of the endosperm. AtCSLA7 is widely expressed, and is likely to be required for synthesis of a cell wall polysaccharide found throughout the plant. Our results suggest that this polysaccharide is essential for cell wall structure or for signaling during plant embryo development. PMID:12586879

Data on partial polyhydroxyalkanoate synthase genes (phaC) mined from Aaptos aaptos marine sponge-associated bacteria metagenome.

PubMed

Amelia, Tan Suet May; Amirul, Al-Ashraf Abdullah; Bhubalan, Kesaven

2018-02-01

We report data associated with the identification of three polyhydroxyalkanoate synthase genes (phaC) isolated from the marine bacteria metagenome of Aaptos aaptos marine sponge in the waters of Bidong Island, Terengganu, Malaysia. Our data describe the extraction of bacterial metagenome from sponge tissue, measurement of purity and concentration of extracted metagenome, polymerase chain reaction (PCR)-mediated amplification using degenerate primers targeting Class I and II phaC genes, sequencing at First BASE Laboratories Sdn Bhd, and phylogenetic analysis of identified and known phaC genes. The partial nucleotide sequences were aligned, refined, compared with the Basic Local Alignment Search Tool (BLAST) databases, and released online in GenBank. The data include the identified partial putative phaC and their GenBank accession numbers, which are Rhodocista sp. phaC (MF457754), Pseudomonas sp. phaC (MF437016), and an uncultured bacterium AR5-9d_16 phaC (MF457753).

Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes

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

Armstrong, G.A.; Hearst, J.E.; Alberti, M.

1990-12-01

Carotenoids comprise one of the most widespread classes of pigments found in nature. The first reactions of C{sub 40} carotenoid biosynthesis proceed through common intermediates in all organisms, suggesting the evolutionary conservation of early enzymes from this pathway. The authors report here the nucleotide sequence of three genes from the carotenoid biosynthesis gene cluster of Erwinia herbicola, a nonphotosynthetic epiphytic bacterium, which encode homologs of the CrtB, CrtE, and CrtI proteins of Rhodobacter capsulatus, a purple nonsulfur photosynthetic bacterium. CrtB (prephytoene pyrophosphate synthase), CrtE (phytoene synthase), and CrtI (phytoene dehydrogenase) are required for the first three reactions specific to themore » carotenoid branch of general isoprenoid metabolism. All three dehydrogenases possess a hydrophobic N-terminal domain containing a putative ADP-binding {beta}{alpha}{beta} fold characteristic of enzymes known to bind FAD or NAD(P) cofactors. These data indicate the structural conservation of early carotenoid biosynthesis enzymes in evolutionary diverse organisms.« less

Microbial-type terpene synthase genes occur widely in nonseed land plants, but not in seed plants

DOE PAGES

Jia, Qidong; Li, Guanglin; Köllner, Tobias G.; ...

2016-10-10

Here, the vast abundance of terpene natural products in nature is due to enzymes known as terpene synthases (TPSs) that convert acyclic prenyl diphosphate precursors into a multitude of cyclic and acyclic carbon skeletons. Yet the evolution of TPSs is not well understood at higher levels of classification. Microbial TPSs from bacteria and fungi are only distantly related to typical plant TPSs, whereas genes similar to microbial TPS genes have been recently identified in the lycophyte Selaginella moellendorffii. The goal of this study was to investigate the distribution, evolution, and biochemical functions of microbial terpene synthase-like ( MTPSL) genes inmore » other plants. By analyzing the transcriptomes of 1,103 plant species ranging from green algae to flowering plants, putative MTPSL genes were identified predominantly from nonseed plants, including liverworts, mosses, hornworts, lycophytes, and monilophytes. Directed searching for MTPSL genes in the sequenced genomes of a wide range of seed plants confirmed their general absence in this group. Among themselves, MTPSL proteins from nonseed plants form four major groups, with two of these more closely related to bacterial TPSs and the other two to fungal TPSs. Two of the four groups contain a canonical aspartate-rich “DDxxD” motif. The third group has a “DDxxxD” motif, and the fourth group has only the first two “DD” conserved in this motif. Upon heterologous expression, representative members from each of the four groups displayed diverse catalytic functions as monoterpene and sesquiterpene synthases, suggesting these are important for terpene formation in nonseed plants.« less

Microbial-type terpene synthase genes occur widely in nonseed land plants, but not in seed plants

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

Jia, Qidong; Li, Guanglin; Köllner, Tobias G.

Here, the vast abundance of terpene natural products in nature is due to enzymes known as terpene synthases (TPSs) that convert acyclic prenyl diphosphate precursors into a multitude of cyclic and acyclic carbon skeletons. Yet the evolution of TPSs is not well understood at higher levels of classification. Microbial TPSs from bacteria and fungi are only distantly related to typical plant TPSs, whereas genes similar to microbial TPS genes have been recently identified in the lycophyte Selaginella moellendorffii. The goal of this study was to investigate the distribution, evolution, and biochemical functions of microbial terpene synthase-like ( MTPSL) genes inmore » other plants. By analyzing the transcriptomes of 1,103 plant species ranging from green algae to flowering plants, putative MTPSL genes were identified predominantly from nonseed plants, including liverworts, mosses, hornworts, lycophytes, and monilophytes. Directed searching for MTPSL genes in the sequenced genomes of a wide range of seed plants confirmed their general absence in this group. Among themselves, MTPSL proteins from nonseed plants form four major groups, with two of these more closely related to bacterial TPSs and the other two to fungal TPSs. Two of the four groups contain a canonical aspartate-rich “DDxxD” motif. The third group has a “DDxxxD” motif, and the fourth group has only the first two “DD” conserved in this motif. Upon heterologous expression, representative members from each of the four groups displayed diverse catalytic functions as monoterpene and sesquiterpene synthases, suggesting these are important for terpene formation in nonseed plants.« less