Increased carotenoid content of Xanthophyllomyces dendrorhous cultivated in plant oil supplemented media

Carotenoid pigments (particularly astaxanthin) of the red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) have economical importance as food and feed colouring additives. Application of nutrients stimulating astaxanthin synthesis would improve the pigment production of the fungus. Vegetable oils contain various unsaturated fatty acids and isoprenoids, among them different precursors of the carotenoid biosynthesis. Th e effect of seven different, commercially available vegetable oils (sesame seed oil, corn seed oil, wheat germ oil, palm oil, pumpkin seed oil, coconut oil and olive oil) on the carotenoid production of two strains representing the teleomorph X. dendrorhous and the anamorph P. rhodozyma was examined. The two strains responded to the presence of the oil additives distinctly. Sesame seed and coconut oil stimulated the pigment production in the X. dendrorhous isolate only, whereas palm oil increased the production of both tested strains

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

Analysis of the transcription and overexpression of the mevalonate-isoprenoid biosynthesis pathway genes in mucor circinelloides

Zygomycetes have a great practical significance in industrial-biotechnological and agricultural fields and also as opportunistic pathogens. Mucor circinelloides is a carotenoid producing filamentous fungus, which has been used as a model organism in various genetic, biochemical and molecular studies. Terpenes are synthesised by a side-route of the general mevalonate-isoprenoid biosynthetic pathway in fungi. Terpene-type metabolites (such as sterols, carotenoids, hormones/pheromones, functional groups of different proteins, e.g. farnesylated or geranylgeranylated proteins) are involved in the formation of the structure of the cell membrane, morphogenesis, electron transport, signal transduction, apoptotic processes, protection against free radicals, cell differentiation, adaptation to environmental changes, etc. Today, ergosterol and its synthesis is a major target of antifungal therapy. Our aim is to reveal the function, regulation of the mevalonate-isoprenoid pathway genes in M. circinelloides. In this study, effects of cultivation time, light, salt stress, media, temperature, oxygen tension, and statin treatment on the transcription of six terpenoid pathway genes, encoding the the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (hmgS), mevalonate kinase (mvk), diphospho-mevalonate decarboxylase (dmd), isopentenyl-pyrophosphate isomerase (ipi), farnesyl-pyrophosphate synthase (isoA) and geranylgeranyl-pyrophosphate synthase (carG), were analysed. The nucleotide sequences of the genes and their regulatory regions, as well as the amino acid sequences of the encoded proteins were analysed. Autonomously replicating vectors, carrying one of the mevalonate-isoprenoid genes under the control of own and Mucor glyceraldehyde-3-phosphate dehydrogenase (gpd1) promoter and terminal sequences were constructed. The promoter of gpd1 is very effective and can be induced by glucose. PEG/CaCl2-mediated protoplast transformation with plasmids harbouring one of the isoprenoid genes (ipi, isoA and carG, respectively) was used to elevate the copy number of the examined genes in M. circinelloides. Viability and germination of spores, morphology, growth intensity and terpene production (e.g. carotenoid and ergosterol) of the resulting transformants were analysed. Investigation of the copy number of the introduced DNA, transcription of the overexpressed genes and effect of the elevated copy numbers on the transcription of the other terpenoid gene are in progress. Árpád Csernetics, Miklós Takó, Gábor Nagy and Csaba Vágvölgyi are supported by TÁMOP 4.2.4. A/2-11-1-2012-0001 „National Excellence Program – Elaborating and operating an inland student and researcher personal support system convergence program” The project was subsidized by the European Union and co-financed by the European Social Fund. The infrastructure and research equipment was supported by TÁMOP4.1.1.C- 12/1/KONV-2012-0014 and OTKA NN 106394

Expression of a bacterial β-carotene hydroxylase in canthaxanthin producing mutant Mucor circinelloides strains

Xanthophylls, primarily hydroxyl- and keto-derivatives of β-carotene (such as zeaxanthin, β-cryptoxanthin, canthaxanthin, echinenone and astaxanthin) have powerful antioxidant activity. Due to several positive effects on human and animal health, industrial application of xanthophylls shows an increasing tendency. In our previous study, carotenoid biosynthesis of the β-carotene producing zygomycetes, Mucor circinelloides was modified by integrating the β-carotene ketolase encoding gene (crtW) of Paracoccus sp. N81106 into the genome. The isolated mutants accumulated mainly canthaxanthin and echinenone. Although, the fungus has β-carotene hydroxylase activity the astaxanthin production remained low even under different cultivation conditions, whereby canthaxanthin accumulation was further enhanced. In this study, the β-carotene hydroxylase encoding gene (crtZ) of the same bacterium was introduced into these canthaxanthin producing mutants of M. circinelloides either on autonomously replicating plasmids or by integrating it into the genome using the REMI method. Increased astaxanthin accumulation was observed in the transformants, which together with zeaxanthin and β-cryptoxanthin content could be further enhanced several fold by the modification of the cultivation conditions. An effective method for the extraction of carotenoids from Mucor mycelia grown in liquid culture was also developed

Modification of the carotenoid production of mucor circinelloides using different xanthophyllomyces dendrorhous crts genes

Canthaxanthin and astaxanthin are oxygenated derivatives of β-carotene and have several beneficial effects on the animal and human health. They are mainly used as feed additives, especially for fishes and poultry. The aim of the present study is to examine the biological requirements for the microbial production of these natural pigments. Mucor circinelloides has been used as model organism of the microbial carotenoid biosynthesis. Previously, canthaxanthin production was achieved in this fungus by integrating a modified bacterial β-carotene-ketolase gene in the genome of M. circinelloides. In the present study, crtS genes of different X. dendrorhous strains were cloned and expressed in β-carotene producing wild type and canthaxanthin producing mutant M. circinelloides strains. X. dendrorhous is an astaxanthin producing basidiomycete yeast. Previously, it was proven, that the cythochrome-P450 hydroxylase enzyme encoded by the crtS gene has β-carotenehydroxylase activity (ÁLVAREZ et al., 2006). A hypothethical β-carotene-ketolase activity has also been suggested (OJIMA et al., 2006), but it has not been verified yet. In our study, crtS genes with different sequences were amplified and several plasmids were constructed carrying the isolated genes under the control of the regulator regions of the Mucor glyceraldehyde-3-phosphate dehydrogenase 1 gene. The plasmids were transformed into the fungal protoplasts. Analysis of the carotenoid content of the transformants revealed astaxanthin production in the resulting strains. This work has been supported by the KTIA-OTKA grant CK 80188