Chemical investigation of novel ascomycetes using PCR based screening approaches
Fungi are well known for a wealth of pharmacologically important activities and agrochemical properties. Polyketides that
are widely found in fungi, are a large group of secondary metabolites which exhibit diversity in their function and structure.
Here we described an investigation of three fungal strains which were prospected for production of polyketides. The aim of
this work was to employ the diversity of reducing type I polyketide synthase genes in these fungi using a molecular and bioinformatics
approaches as a mini tool. A degenerate primer pair for highly reduced PKSs was newly designed and used together with ketosynthase
primers for amplification. One hundred and thirty-eight clones were sequenced. Ten KS domain sequences were isolated, using
two primer pairs specific for highly reduced type PKSs. This study revealed four sequences from Emarcea castanopsidicola, four ketosynthase sequences from Gaeumannomyces amomi and two sequences from Leiosphaerella amomi, respectively. Bioinformatic techniques were employed to identify a group of these KS domain sequences. Based on these sequences
suggested that rapid screening provided the potential to explore significant PKS structural diversity. Hence chemical investigation
had been conducted and exhibited nine compounds. The endophytic fungus L. amomi was cultivated and elucidated linoleic acid, ergosterol and an unidentified sterol in the extracts. Linoleic acid, sitosterol,
and p-hydroxybenzoic acid were isolated from the saprobic fungus E. castanopsidicola. We first isolated a new polyketide, stemphol 1-O-β-D-galactopyranoside together with four known metabolites; stemphol, kojic acid, ergosterol, indole-3-carboxylic acid from
an ethyl acetate extract of the cultures of G. amomi. Stemphol was classified as a phenolic lipid or resorcinolic lipid, which have biopharmacological, biomedical, and biotechnological
importance. However, recent researches have revealed that these molecule types are synthesized by 2′-oxoalkylresorcinolic
acid synthase. The prospective KS domain sequences from this study will be used as probes to isolate putative PKS genes. A
gene cluster responsible for PK biosynthesis should be confirmed by determination of PK products generated by these enzymes.
–Polyketide synthase–Ketosynthase domain–Endophytic fungi–Stemphol 1-O-β-D-galactopyranoside
Available from: Nikolas Fokialakis
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ABSTRACT: Mushrooms comprise a vast source of new pharmaceutical and nutraceutical products. Submerged fermentation of the mycelial form of mushroom-producing fungi is promising for efficient production of their biomass and active metabolites. The aim of this study was the isolation and identification of bioactive metabolites derived from the edible mushroom Pleurotus ostreatus biomass, produced by submerged fermentation in a batch stirred tank bioreactor. The dichloromethane extract was fractioned by medium pressure liquid chromatography (MPLC) and Sephadex LH-20 column chromatography. For extracting efficiently the phenolic compounds of the methanolic extract, an adsorption-desorption process, using XAD-4 type resin, was performed. The pure compounds were elucidated with 1D/2D NMR-spectroscopic analyses, NMR data comparisons, and chemical correlations combined with GC/MS-LC/MS experiments. The compounds afforded by the dichloromethane extract were identified as linoleic acid (1), oleic acid (2), stearic acid (3), palmitic acid (4) and their corresponding methyl esters (5-8, respectively), benzoic acid (9), trans 3, 4-dihydro-3, 4, 8-trihydroxynapthalen-1(2H)-one (10), 4-hydroxybenzaldehyde (11), indolo-3-carboxylic acid (12) and uracil (13). The investigation of the methanolic extract led to the isolation of 3-formyl-pyrrole (14), 4-hydroxy-benzoic acid (15), uridine (16), nicotinic acid (17) and nicotinamide (18). Based on existing literature data, all these compounds exhibit valuable biological properties. The information obtained is considered fundamental for further investigation of the P. ostreatus fermentation process on an industrial scale for enhanced bioactive metabolite production. (c) 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of 11th International Congress on Engineering and Food (ICEF 11) Executive Committee.
12/2011; 1:1746-1752. DOI:10.1016/j.profoo.2011.09.257
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ABSTRACT: Covering: 2007 to 2011. Previous review: Nat. Prod. Rep., 2007, 24, 750Common to all FASs, PKSs and NRPSs is a remarkable component, the acyl or peptidyl carrier protein (A/PCP). These take the form of small individual proteins in type II systems or discrete folded domains in the multi-domain type I systems and are characterized by a fold consisting of three major α-helices and between 60-100 amino acids. This protein is central to these biosynthetic systems and it must bind and transport a wide variety of functionalized ligands as well as mediate numerous protein-protein interactions, all of which contribute to efficient enzyme turnover. This review covers the structural and biochemical characterization of carrier proteins, as well as assessing their interactions with different ligands, and other synthase components. Finally, their role as an emerging tool in biotechnology is discussed.
Natural Product Reports 08/2012; 29(10):1111-37. DOI:10.1039/c2np20062g · 10.11 Impact Factor
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ABSTRACT: The aim of this study was to investigate the potential of the submerged fermentation procedure in the production of bioactive metabolites of the common edible mushroom Pleurotus ostreatus. The biomass of the mushroom strain was produced by submerged fermentation in a batch stirred tank bioreactor and extracted by solvents of increasing polarity. The dichloromethane and methanol extract were fractioned by different techniques including Adsorption Chromatography and Fast Centrifugal Partition Chromatography (FCPC). The structures of pure compounds were elucidated with 1D/2D NMR-spectroscopic analyses, and chemical correlations combined with GC/MS and LC/MS experiments. Nineteen metabolites (e.g., fatty acids, phenolic metabolites, nucleotides and alkaloids) were isolated. Beyond the production of known metabolites, we report herein the production also of trans-3,4-dihydro-3,4,8-trihydroxynapthalen-1(2H)-one, indolo-3-carboxylic acid, 3-formylpyrrole and 4-hydroxybenzoic acid, that have pharmaceutical interest and are isolated for the first time from Pleurotus strains. This work indicates the great potential of the established bioprocess for the production of P. ostreatus mycelia with enhanced metabolic profile.
Molecules 12/2012; 17(3):2714-24. DOI:10.3390/molecules17032714 · 2.42 Impact Factor
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