Effect of different statins on the antifungal activity of polyene antimycotics

The antifungal activities of amphotericin B/statin and nystatin/statin combinations against some opportunistic pathogenic fungi (Candida albicans, Candida glabrata, Paecilomyces variotii, Aspergillus fumigatus, Aspergillus flavus and Rhizopus oryzae) were investigated. The in vitro interactions between polyene antifungal drugs and different statins were evaluated using a standard chequerboard broth microdilution method. Most of the detected interactions were additive, though in some cases synergism was also observed. In most cases, the extents of inhibition were higher when these compounds were applied together, and as a result the concentrations of amphotericin B and a given statin, needed to prevent fungal growth, generally could be decreased by some dilution steps

Astaxanthin bioszintézis genetikai befolyásolása Phaffia rhodozyma-ban = Genetic influence on biosynthesis of astaxanthin in Phaffia rhodozyma

Az astaxanthin (3,3¢-dihydroxy-b,b-carotene-4,4¢-dione) termelő élesztők nagy biotechnológiai jelentőségűek. RAPD analízisen alapuló csoport analízist végrehajtva igazoltuk , hogy az astaxanthin-termelő izolátumok két fajba (Xanthopyllomyces dendrorhous és Phaffia rhodozyma) tartoznak. Különböző mutációs eljárásokat - gamma-, UV-sugárzás és kémiai mutagenezis, illetve ezek kombinációi - alkalmazva auxotróf és színmutánsokat izoláltunk mindkét élesztőfajból. Pigment- illetve astaxanthin túltermelő mutánsokat, melyek színanyag profilja jelentősen eltért hoztunk létre. Protoplaszt fúziós kísérleteket végeztünk és fúziós hibrideket izoláltunk és jellemeztünk. In vivo EPR vizsgálatokkal jellemeztük a különböző pigment-profilú mutánsok plazmamembránjának szerkezeti és dinamikus sajátosságait a hőmérséklettel összefüggésben. Meghatároztuk a X. dendrorhous 3-hydroxy-3-methylglutharyl coenzyme A reductase (HMG-CoA reductase) génjének részleges génszekvenciáját. Felfedeztük, hogy mind a P. rhodozyma és a X. dendrorhous képes lebontani az ochratoxin A-t ochratoxin alfává, feltehetően egy sejthez kötött karboxipeptidáz A aktivitás révén. | Astaxanthin (3,3¢-dihydroxy-b,b-carotene-4,4¢-dione) - accumulating yeasts are of great biotechnological interest. RAPD analysis involving astaxanthin-producing yeast strains was performed: cluster analysis based on RAPD markers differentiated isolates of Xanthopyllomyces dendrorhous and Phaffia rhodozyma (asexual) at an intraspecific level. Different mutation strategies, which involves *- and UV-irradiations, as well as chemical mutagenesis of cells was worked out to obtain auxotrophic and colour mutants from these astaxanthin-producing yeasts. Overproducer mutants exhibited different patterns of carotenoid production have also been isolated. Protoplast fusion experiment have been carried out and fusion hybrides have been isolated and characterized. Spin labelling methods were used to describe the structure and dynamic properties of various carotenoids containing X. dendrorhous’s plasma membranes in relation to temperature. A partial gene sequence of 3-hydroxy-3-methylglutharyl coenzyme A reductase (HMG-CoA reductase) of X. dendrorhous has been determined. Ochratoxin degrading activities of P. rhodozyma and X. dendrorhous isolates were discovered. P. rhodozyma is able to convert ochratoxin A to ochratoxin a (possibly mediated by a cell-bound carboxypeptidase A)

A karotinoid termelés genetikai hátterének vizsgálata genetikailag módosított járomspórás gombák segítségével. = Molecular study of the cartenoid biosynthetic pathway using genetically modified zygomycetous fungal strains

A pályázati program célja, hogy rekombináns technikákat, valamint a genetikai transzformáció különféle eljárásait alkalmazva járomspórás gombák karotinoid bioszintézisét módosítsuk, továbbá, hogy az így létrehozott rendszerben vizsgáljuk a bioszintetikus út lefolyását és a produkció mértékét. Célunk volt, hogy megvizsgáljuk a b-karotinból kiinduló asztaxantin szintézis lehetőségét Mucor circinelloidesben, valamint karotintúltermelő mutánsok előállítása. Mintegy húsz módosult karotinösszetételű (asztaxantin és/vagy kantaxantin termelésre képes), illetve karotin túltermelő M. circinelloides törzset állítottunk elő és jellemeztünk részletesen. Azonosítottuk és részletesen jellemeztük a Rhizomucor miehei HMG-KoA reduktáz, valamint a M. circinelloides izopentenil pirofoszfát izomeráz génjét. Nagyszámú, a karotintermelés módosítását lehetővé tevő transzformációs vektort készítettünk, melyek a további törzsnemesítésben felhasználhatók. 20 járomspórás gombafaj különböző, vad típusú törzseinek karotintermelését elemeztük, különböző környezeti feltételek mellett. Több nagy karotinoid produkcióra képes, illetve új karotinösszetételű törzset azonosítottunk, melyek a további munkákban felhasználhatók. Új, a későbbi alap- és alkalmazott kutatásokban is alkalmazható eljárásokat, protokolokat dolgoztunk ki elsősorban a járomspórás gombák genetikai tramszformációjára, a karotin termeltetés körülményeire (tenyésztés, fermentáció) és a termelés fokozására vonatkozóan. | The objectives of the project were the followings: development of carotene superproducing strains by means of the over-expression of the mevalonate-isoprenoid pathway; introduction of heterologous genes into Mucor circinelloides in order to achieve the production of oxygenated b-carotene derivatives; and extension of these studies to other carotene producer Zygomycetes. Isopentenyl pyrophosphate isomerase gene (McIPP) of Mucor encoding a key-enzyme of the isoprenoid pathway was cloned and analysed. For the modification of the mevalonate pathway, functional analysis of the HMG-CoA reductase gene (hmg) of Rhizomucor miehei was completed and transforming vectors were constructed. Transformation experiments with plasmids harbouring the genes: hmg; isoA (FPP synthase); carG (GGPP synthase) and McIPP, were carried out and several strains with elevated carotene production were obtained. The crtW and crtZ genes from Agrobacterium aurantiacum were placed under the control of M. circinelloides expression signals and heterologous transformations were performed on a selected M. circinelloides strain. Transformants able to produce astaxanthin, cantaxanthin and/or zeaxanthin and β-cryptoxanthin were isolated. Agrobacterium tumefaciens-mediated transformation systems allowing integrative transformation were elaborated and optimized for M. circinelloides and other related carotenoid producer species (other Mucor, Gilbertella and Backusella species)

A Mortierellales rend (Fungi, Zygomycetes) átfogó filogenetikai és taxonómiai revíziója morfológiai, fiziológiai és molekuláris eljárásokkal = Reconstruction of the phylogeny of the filamentous fungal order Mortierellales (Zygomycetes): a comprehensive taxonomical revision

A projekt célja a Mortierellales fonalas gomba rend átfogó filogenetikai és taxonómiai revíziója volt. E lipid akkumuláló szervezetek nagy jelentőséggel rendelkeznek, mint többszörösen telítetlen zsírsavak termelői és különböző vegyületek biotranszformáló ágensei. A projekt során létrehoztunk egy több, mint 200 törzsből álló gyűjteményt, mely a gombacsoport minden tenyészthető nemzetségét felöleli és mintegy 100 faj típustörzsét is tartalmazza. Az elhelyezett törzsek esetében meghatároztuk a teljes ITS régió, valamint a 18S és a 28S rRNS gén szekvenciáit és ezek alapján filogenetikai elemzést végeztünk. Több mint száz törzs esetében meghatároztuk a tef és RPB1 fehérjekódoló gének egy szakaszát is és az így létrehozott ötgénes adatsor alapján egy teljesebb filogenetikai elemzést végeztünk. A multigén-alapú törzsfák alapján megállapítottuk, hogy a jelenlegi morfológiai alapon definiált Mortierella nemzetség parafiletikus és meghatároztuk a renden belüli monofiletikus fajcsoportokat, egyszersmind egy új, filogenetikai szempontból megfelelőbb rendszerezés alapjait is leraktuk. Az így meghatározott csoportok morfológiai és fiziológiai jellemzését is elvégeztük. A Mortierellales rend részletes elemzése értékes információt szolgáltatott a későbbi bioaktív metabolitok és enzimaktivitások felfedezését célzó alap- és gyakorlati kutatások számára. Ugyanakkor, a nagy szekvencia, morfológiai és fiziológiai adatsorok elemzése alapján tágabb evolúciós következtetéseket is le tudtunk vonni. | The aim of the present project was a comprehensive phylogenetic and taxonomic revision of the fungal order, Mortierellales. These lipid-accumulating organisms have great importance as producers of polyunsaturated fatty acids or biotransforming agents of several chemical compounds. In this project, we established an outstanding strain collection consisting of more than 200 strains. It covers all cultivable genera of the order and includes approximately 100 type strains. Sequences of the complete ITS region and the SSU and LSU RNA genes were determined in case of the deposited strains and were used for phylogenetic analyses. In case of more than 100 strains, the protein coding tef and RPB1 genes were also sequenced and the resulting five-gene dataset was used in a comprehensive phylogenetic analysis. These data showed the genus Mortierella to be paraphyletic. Based on the multigene phylogenies, we proposed new monophyletic species-groups within the order and established the bases of a new phylogenetically correct taxonomy. Besides the molecular analyses, morphological and physiological examination of the newly determined groups was also performed. The detailed characterization of Mortierellales served valuable new information for further studies with the aim of discovering new bioactive metabolites and enzyme activities. At the same time, analysis of the large molecular, morphological and physiological data sets allowed us to examine evolutional problems in a wider context

Molecular Characterization of Novel Mycoviruses in Seven Umbelopsis Strains

The presence of viruses is less explored in Mucoromycota as compared to other fungal groups such as Ascomycota and Basidiomycota. Recently, more and more mycoviruses are identified from the early-diverging lineages of fungi. We have determined the genome of 11 novel dsRNA viruses in seven different Umbelopsis strains with next-generation sequencing (NGS). The identified viruses were named Umbelopsis ramanniana virus 5 (UrV5), 6a (UrV6a); 6b (UrV6b); 7 (UrV7); 8a (UrV8a); 8b (UrV8b); Umbelopsis gibberispora virus 1 (UgV1); 2 (UgV2) and Umbelopsis dimorpha virus 1a (UdV1a), 1b (UdV1b) and 2 (UdV2). All the newly identified viruses contain two open reading frames (ORFs), which putatively encode the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively. Based on the phylogeny inferred from the RdRp sequences, eight viruses (UrV7, UrV8a, UrV8b, UgV1, UgV2, UdV1a, UdV1b and UdV2) belong to the genus Totivirus, while UrV5, UrV6a and UrV6b are placed into a yet unclassified but well-defined Totiviridae-related group. In UrV5, UgV1, UgV2, UrV8b, UdV1a, UdV2 and UdV1b, ORF2 is predicted to be translated as a fusion protein via a rare +1 (or-2) ribosomal frameshift, which is not characteristic to most members of the Totivirus genus. Virus particles 31 to 32 nm in diameter could be detected in the examined fungal strains by transmission electron microscopy. Through the identification and characterization of new viruses of Mucoromycota fungi, we can gain insight into the diversity of mycoviruses, as well as into their phylogeny and genome organization

Hygromycin B, carboxin and nourseothricin susceptibility of polyunsaturated fatty acid producing Mortierella and Umbelopsis strains

Mortierella and Umbelopsis species are particularly active in polyunsaturated fatty acid (PUFA) synthesis as they are able to produce many ω-3 and ω-6 PUFAs. Genetic manipulation of the lipid production to generate PUFA overproducing strains and strains with altered PUFA profile requires well-established transformation systems and reliable selectable markers. Therefore, we screened different antifungal agents, which can be used for selection in further transformation experiments. Hygromycin B, carboxin, pyrithiamine and nourseothricin susceptibility of several Mortierella and Umbelopsis isolates was investigated using a broth microdilution method. Pyrithiamine was totally ineffective against all isolates while the other three antifungal agents were active against Mortierella and Umbelopsis strains. Several Mortierella isolates represented high sensitivity to hygromycin B whilst nourseothricin was rather active against Umbelopsis species. Carboxin inhibited the hyphal growth and the spore germination of all isolates completely in low concentrations

Data Partitions, Bayesian Analysis and Phylogeny of the Zygomycetous Fungal Family Mortierellaceae, Inferred from Nuclear Ribosomal DNA Sequences

Although the fungal order Mortierellales constitutes one of the largest classical groups of Zygomycota, its phylogeny is poorly understood and no modern taxonomic revision is currently available. In the present study, 90 type and reference strains were used to infer a comprehensive phylogeny of Mortierellales from the sequence data of the complete ITS region and the LSU and SSU genes with a special attention to the monophyly of the genus Mortierella. Out of 15 alternative partitioning strategies compared on the basis of Bayes factors, the one with the highest number of partitions was found optimal (with mixture models yielding the best likelihood and tree length values), implying a higher complexity of evolutionary patterns in the ribosomal genes than generally recognized. Modeling the ITS1, 5.8S, and ITS2, loci separately improved model fit significantly as compared to treating all as one and the same partition. Further, within-partition mixture models suggests that not only the SSU, LSU and ITS regions evolve under qualitatively and/or quantitatively different constraints, but that significant heterogeneity can be found within these loci also. The phylogenetic analysis indicated that the genus Mortierella is paraphyletic with respect to the genera Dissophora, Gamsiella and Lobosporangium and the resulting phylogeny contradict previous, morphology-based sectional classification of Mortierella. Based on tree structure and phenotypic traits, we recognize 12 major clades, for which we attempt to summarize phenotypic similarities. M. longicollis is closely related to the outgroup taxon Rhizopus oryzae, suggesting that it belongs to the Mucorales. Our results demonstrate that traits used in previous classifications of the Mortierellales are highly homoplastic and that the Mortierellales is in a need of a reclassification, where new, phylogenetically informative phenotypic traits should be identified, with molecular phylogenies playing a decisive role

Molecular Characterization of Novel Mycoviruses in Seven Umbelopsis Strains

The presence of viruses is less explored in Mucoromycota as compared to other fungal groups such as Ascomycota and Basidiomycota. Recently, more and more mycoviruses are identified from the early-diverging lineages of fungi. We have determined the genome of 11 novel dsRNA viruses in seven different Umbelopsis strains with next-generation sequencing (NGS). The identified viruses were named Umbelopsis ramanniana virus 5 (UrV5), 6a (UrV6a); 6b (UrV6b); 7 (UrV7); 8a (UrV8a); 8b (UrV8b); Umbelopsis gibberispora virus 1 (UgV1); 2 (UgV2) and Umbelopsis dimorpha virus 1a (UdV1a), 1b (UdV1b) and 2 (UdV2). All the newly identified viruses contain two open reading frames (ORFs), which putatively encode the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively. Based on the phylogeny inferred from the RdRp sequences, eight viruses (UrV7, UrV8a, UrV8b, UgV1, UgV2, UdV1a, UdV1b and UdV2) belong to the genus Totivirus, while UrV5, UrV6a and UrV6b are placed into a yet unclassified but well-defined Totiviridae-related group. In UrV5, UgV1, UgV2, UrV8b, UdV1a, UdV2 and UdV1b, ORF2 is predicted to be translated as a fusion protein via a rare +1 (or-2) ribosomal frameshift, which is not characteristic to most members of the Totivirus genus. Virus particles 31 to 32 nm in diameter could be detected in the examined fungal strains by transmission electron microscopy. Through the identification and characterization of new viruses of Mucoromycota fungi, we can gain insight into the diversity of mycoviruses, as well as into their phylogeny and genome organization