DE102004047409A1 - Use of laccases as reporter genes - Google Patents
Use of laccases as reporter genes Download PDFInfo
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- DE102004047409A1 DE102004047409A1 DE200410047409 DE102004047409A DE102004047409A1 DE 102004047409 A1 DE102004047409 A1 DE 102004047409A1 DE 200410047409 DE200410047409 DE 200410047409 DE 102004047409 A DE102004047409 A DE 102004047409A DE 102004047409 A1 DE102004047409 A1 DE 102004047409A1
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- laccase
- dna
- sequence
- candida
- expression
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- 241000191353 [Candida] haemulonis Species 0.000 description 1
- 241000192319 [Candida] insectorum Species 0.000 description 1
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- LHGVFZTZFXWLCP-UHFFFAOYSA-N guaiacol Chemical class COC1=CC=CC=C1O LHGVFZTZFXWLCP-UHFFFAOYSA-N 0.000 description 1
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- 238000011835 investigation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
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- 229920005610 lignin Polymers 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 description 1
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- 238000010369 molecular cloning Methods 0.000 description 1
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- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
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- 229930003231 vitamin Natural products 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0393—Animal model comprising a reporter system for screening tests
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Verfahren zur Bestimmung von regulatorischen Eigenschaften eines genetischen Elementes, das die Schritte umfasst: DOLLAR A (a) Einführen eines DNA-Moleküls mit einer DNA-I-Sequenz, deren regulatorische Eigenschaften bestimmt werden sollen, in eine Zelle, wobei die DNA-I-Sequenz mit einer weiteren DNA-II-Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, so verknüpft ist, dass die regulatorischen Elemente der DNA-I-Sequenz die Expression der Laccase kontrollieren, DOLLAR A (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression der Laccase erlauben, DOLLAR A (c) Zuordnen der unter (b) gewählten Bedingungen zu den regulatorischen Eigenschaften der DNA-I-Sequenz.A method of determining regulatory properties of a genetic element comprising the steps of: DOLLAR A (a) introducing into a cell a DNA molecule having a DNA I sequence whose regulatory properties are to be determined, wherein the DNA Sequence is linked to another DNA II sequence coding for an Aspergillus nidulans laccase in such a way that the regulatory elements of the DNA-I sequence control the expression of the laccase, DOLLAR A (b) growing under (a) cells under conditions which allow the expression of the laccase, DOLLAR A (c) assigning the conditions chosen under (b) to the regulatory properties of the DNA-I sequence.
Description
Die Erfindung betrifft die Verwendung von Laccasen aus Aspergillus nidulans als Reportersysteme in Verfahren zur qualitativen und quantitativen Bestimmung von Genexpression, insbesondere zur qualitativen und quantitativen Bestimmung von Promotorstärken, in Verfahen zur Lokalisation von Proteinen in einer Zelle, sowie Verfahren zur Selektion bestimmter rekombinanter Zellen sowie in Verfahren zur heterologen Genexpression.The This invention relates to the use of laccases from Aspergillus nidulans as reporter systems in qualitative and quantitative processes Determination of gene expression, especially for qualitative and quantitative determination of promoter strengths, in process of localization of proteins in a cell, as well as methods for the selection of certain recombinant cells as well as in methods for heterologous gene expression.
Laccasen (EC 1.10.3.2) sind blaue Multi-Kupferenzyme, die von Pilzen und Pflanzen produziert werden. Sie oxidieren eine Reihe aromatischer Verbindungen wie Mono-, Di, und Polyphenole, aromatische Amine und Methoxy-phenole. Bei der Oxidation werden dem Substrat Elektronen entzogen, im Enzym "gesammelt" und dann jeweils vier Elektronen auf Sauerstoff übertragen und Wasser gebildet. (Messerschmidt & Huber, 1990).laccases (EC 1.10.3.2) are blue multi-hybrid enzymes, those of fungi and Plants are produced. They oxidize a number of aromatic ones Compounds such as mono-, di-, and polyphenols, aromatic amines and Methoxy-phenols. During oxidation, the substrate becomes electrons withdrawn, "collected" in the enzyme and then each transfer four electrons to oxygen and water formed. (Messerschmidt & Huber, 1990).
Pilze enthalten in der Regel mehrere Laccasen, die vermutlich unterschiedliche biologische Funktionen ausüben. Im Basidiomycenten Coprinus cinereus wurden acht Laccasen beschrieben, während im Ascomyceten A. nidulans sechs Laccasen vorkommen (Wagner, 2003; Hoegger et al., 2004). Die genaue biologische Rolle der pilzlichen Laccasen ist weitgehend unbekannt. Sie besitzen meistens ein N-terminales Sekretionssignal und werden sekretiert. Am besten ist die Funktion der Basidiomycetenlaccasen im Ligninabbau untersucht (Crestini et al., 2003). In anderen Pilzen wird eine Funktion bei der Melaninbiosynthese angenommen oder beim Abbau von phenolischen Substanzen (Hermann et al., 1983; Fernández-Larrea & Stahl, 1996).mushrooms usually contain several laccases, which are probably different exercise biological functions. In the Basidiomycenten Coprinus cinereus eight laccases have been described, while in the Ascomycete A. nidulans six laccases occur (Wagner, 2003; Hoegger et al., 2004). The exact biological role of the fungal Laccases is largely unknown. They usually have an N-terminal Secretion signal and are secreted. The best is the function of basidiomycete laccases in lignin degradation (Crestini et al., 2003). In other mushrooms, a function in melanin biosynthesis assumed or in the degradation of phenolic substances (Hermann et al., 1983; Fernández-Larrea & Stahl, 1996).
Reportergene wie beta-Galaktosidase oder Glucuronidase werden vielfältig in der Molekularbiologie und Biotechnologie eingesetzt. Sie haben allerdings den Nachteil, dass die Substrate in die Zellen eingebracht werden müssen. Ausserdem besitzen viele Pilze endogene Enzyme, die zu hohen basalen Enzymaktivitäten führen.reporter Gene Such as beta-galactosidase or glucuronidase become diverse in molecular biology and biotechnology. You have, however the disadvantage that the substrates are introduced into the cells have to. In addition, many fungi have endogenous enzymes that are high in basal levels Lead enzyme activities.
Weitere
häufig
verwendete Reportergene stellen die fluoreszierenden Proteine (Green
Fluorescent Protein GFP, und Yellow Fluorescent Protein, zur Ausbildung
der Fluoreszenz und werden vielfältig
zur Lokalisierung von Proteinen aber auch zur Messung von Promotorstärken verwendet
(
Es bestand daher die Aufgabe, neue Verfahren für genetische Screeningverfahren wie Nachweis der Expression eines bestimmten Gens, Messung der Expressionsstärke eines genetischen Regulationssignals, Nachweis der Lokalisation eines Proteins in der Zelle (Zellkompartimenten), bereitzustellen, die nicht die o.g. die o.g. Restriktionen und Nachteile aufweisen.It It was therefore the task of new methods for genetic screening such as detection of the expression of a particular gene, measurement of the expression level of a genetic regulation signal, detection of the localization of a Protein in the cell (cell compartments), which provide not the o.g. the o.g. Have restrictions and disadvantages.
Beschreibung der Erfindungdescription the invention
Die vorliegende Erfindung betrifft in einer ersten Ausführungsform ein Verfahren zur Bestimmung von regulatorischen Eigenschaften eines genetischen Elementes, das die Schritte umfasst:
- (a) Einführen eines DNA-Moleküls mit einer DNA-I-Sequenz, deren regulatorische Eigenschaften bestimmt werden sollen, in eine Zelle, wobei die DNA-I-Sequenz mit einer weiteren DNA-II-Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, so verknüpft ist, dass die regulatorischen Elemente der DNA-I-Sequenz die Expression der Laccase kontrollieren,
- (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression der Laccase erlauben,
- (c) Zuordnen der unter (b) gewählten Bedingungen zu den regulatorischen Eigenschaften der DNA-I-Sequenz.
- (A) introducing into a cell a DNA molecule having a DNA I sequence whose regulatory properties are to be determined, wherein the DNA I sequence is linked to another DNA II sequence coding for a laccase from Aspergillus nidulans is linked so that the regulatory elements of the DNA I sequence control the expression of the laccase,
- (b) culturing the cells obtained under (a) under conditions which allow the expression of the laccase,
- (c) assigning the conditions chosen under (b) to the regulatory properties of the DNA I sequence.
In einer zweiten Ausführungsform betrifft die Erfindung ein Verfahren zur Lokalisation eines gewünschten Proteins in einem Zellkompartiment, das die Schritte umfasst:
- (a) Einführen eines DNA-Moleküls, das eine DNA Sequenz besitzt, die für das gewünschte Protein codiert, und die so mit einer weiteren DNA-II-Sequenz verknüpft ist, die für eine Laccase aus Aspergillus nidulans codiert, das das Protein, das von dem DNA-Molekül codiert wird, eine Fusion aus dem gewünschten Protein mit der Laccase darstellt, in die Zellen,
- (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression des fusionierten Proteins erlauben,
- (c) Nachweis der Lokalisation der Laccase in einem Zellkompartiment und damit der Lokalisation des gewünschten Proteins in diesem Zellkompartiment, insbe sondere durch Zellfraktionierung und anschliessende Bestimmung der Laccaseaktivität in den verschiedenen Fraktionen.
- (a) introducing a DNA molecule having a DNA sequence coding for the desired protein and thus linked to another DNA II sequence coding for an Aspergillus nidulans laccase containing the protein, the is encoded by the DNA molecule, represents a fusion of the desired protein with the laccase into the cells,
- (b) culturing the cells obtained under (a) under conditions which allow the expression of the fused protein,
- (c) Detection of the localization of the laccase in a cell compartment and thus the localization of the desired protein in this cell compartment, in particular by cell fractionation and subsequent determination of laccase activity in the various fractions.
In einer dritten Ausführungsform betrifft die Erfindung ein Verfahren zur Bestimmung der Expression eines gewünschten Gens in einer Zelle, das die Schritte umfasst:
- (a) Einführen eines DNA-Moleküls, das eine DNA Sequenz des gewünschten Gens besitzt, in eine Zelle, wobei die DNA-Sequenz mit einer weiteren DNA-II-Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, so verknüpft ist, dass die regulatorischen Elemente des gewünschten Gens die Expression der Laccase kontrollieren,
- (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression des gewünschten Gens erlauben,
- (c) Nachweis der Expression der Laccase in der Zelle und damit Nachweis der Expression des gewünschten Gens.
- (A) introducing a DNA molecule having a DNA sequence of the desired gene into a cell, wherein the DNA sequence is linked to a further DNA II sequence coding for an Aspergillus nidulans laccase such that the regulatory elements of the desired gene control the expression of the laccase,
- (b) cultivating the cells obtained under (a) under conditions which allow the expression of the desired gene,
- (c) detecting the expression of the laccase in the cell and thus detecting the expression of the desired gene.
In einer vierten Ausführungsform betrifft die Erfindung ein Verfahren zur Expression eines gewünschten Gens in einer Zelle, das die Schritte umfasst:
- (a) Einführen eines DNA-Moleküls, das eine DNA-II-Sequenz, die für eine Laccase aus Aspergillus nidulans codiert, besitzt und mit deren C-terminalem Ende eine weitere DNA-I-Sequenz, die für das gewünschte Genprodukt codiert, so verknüft ist, das die Expression als Fusionsprotein aus Laccase und gewünschtem Genprodukt erfolgt,
- (b) Züchten der unter (a) erhaltenen Zellen unter Bedingungen, die die Expression des gewünschten Gens erlauben,
- (c) Isolierung des Fusionsproteins aus Laccase und gewünschtem Genprodukt.
- (A) introducing a DNA molecule having a DNA II sequence coding for an Aspergillus nidulans laccase, and having at its C-terminal end a further DNA I sequence coding for the desired gene product, as described above linked, the expression takes place as a fusion protein of laccase and the desired gene product,
- (b) cultivating the cells obtained under (a) under conditions which allow the expression of the desired gene,
- (c) isolating the fusion protein from laccase and desired gene product.
Hierbei wird Laccase als Reportergen bzw. Reporterprotein für heterolog exprimierte Proteine verwendet. Viele industriell bedeutende Proteine werden in filamentösen Pilzen produziert. Dabei wird versucht, die Proteine zu sekretieren, um die Aufreinigung zu erleichtern. Laccasen können verwendet werden, um nicht-sekretierte Proteine zu sekretieren. Eine C-terminale Fusion eines gewünschten Proteins mit Laccase führt zur Sekretion des Proteins und ermöglicht, aufgrund der Laccaseaktivität, den leichten Nachweis des sekretierten Proteins. Durch Einführung einer Proteaseschnittstelle zwischen den beiden Proteinen kann das gewünschte Protein freigesetzt werden.in this connection becomes laccase as reporter gene or reporter protein for heterologous used expressed proteins. Many industrially important proteins become filamentous Mushrooms produced. An attempt is made to secrete the proteins, to facilitate the purification. Laccases can be used to non-secreted To secrete proteins. A C-terminal fusion of a desired Protein with laccase leads to secretion of the protein and, due to the laccase activity, allows the light Detection of the secreted protein. By introducing a protease interface between the two proteins, the desired protein can be released become.
Geignete
Zellen für
die o.g. Verfahren sind pflanzliche, beispielsweise Karottenwurzeln,
tierische und menschliche, beispielsweise Hela oder HEK293, und
insbesondere pilzliche Zellen. Besonders gut lassen sich die Verfahren
in Zellen der folgenden Arten ausführen:
Alternaria alternata,Clathrospora
diplospora,Alternaria brassicae,Alternaria brassicicola, Alternaria
raphani, Pleospora herbarum, Pyrenophora trichostoma Cochliobolus
sativus, Cochliobolus heterostrophus, Setosphaeria monoceras Setosphaeria
rostrata, Pleospora betae, Leptosphaeria maculans, Leptosphaeria
microscopica, Ophiobolus herpotrichus, Cucurbitaria elongata,Phoma
sp. Leptosphaeria doliolum,Cucurbitaria berberidis,Paraphaeosphaeria
agavensis Paraphaeosphaeria nolinae,Paraphaeosphaeria obtusispora,Paraphaeosphaeria
conglomerata,Paraphaeosphaeria quadriseptat,Paraphaeosphaeria filamentosa,Melanomma
sanguinarium,Paraphaeosphaeria glauco-punct,Septoria nodorum,Phoma
herbarum,Pleospora rudis,Helminthosporium solani,Helminthosporium
velutinum,Leptosphaeria bicolor,Paraphaeosphaeria michotii,Paraphaeosphaeria
pilleata,Cucurbidothis pityophila,Bimuria novae-zelandiae,Letendraea
helminthicola,Kirschsteiniothelia elaterascu,Helicascus kanaloanus,Herpotrichia
diffusa,Mycosphaerella mycopappi,Herpotrichia juniperi,Byssothecium
circinans,Lophiostoma crenatum,Trematosphaeria heterospora,Kirschsteiniothelia
maritima,Sporomia lignicola,Westerdykella cylindrica,Westerdykella
dispersa, Delitschia didyma,Dendryphiopsis atra,Kirschsteiniothelia
aethiops, Sarcinomyces petricola,Phaeosclera dematioides,Sarcinomyces
crustaceus,Scytalidium dimidiatum,Scytalidium hyalinum,Scytalidium
dimidiatum,Botryosphaeria ribis,Capnobotryella sp.,Capnobotryella
renispora,Coccodinium bartschii,Hortaea werneckii,Phaeotheca fissurella,Acrospermum
compressum ,Acrospermum ramineum,Phaeotrichum benjaminii,Aureobasidium
pullulans,Discosphaerina fagi,Dothidea hippophaeos,Dothidea insculpta,Stylodothis
puccinioides,Dothidea ribesia,Delphinella strobiligena,Myriangium
duriaei,Myriangium duriaei,Piedraia hortae,Raciborskiomyces longisetosum,Capnodium
citri,Euascomycetes sp.,Thecotheus holmskjoldii,Lasallia rossica,Baeomyces
rufus,Anamylopsora pulcherrima,Placopsis gelida,Trapelia involuta,Trapelia
placodioides,Pertusaria trachythallina,Ochrolechia parella,Coccotrema cucurbitula,Lepolichen
coccophorus,Diploschistes rampoddensis,Diploschistes thunbergianus,Graphis
scripta,Conotrema populorum,Gyalecta ulmi,Dibaeis ,Stenocybe pullatula,Sphinctrina
turbinata,Cladonia sulphurina,Cladonia bellidiflora,Cladonia subcervicornis,Metus
conglomeratus,Pilophorus robustus,Pilophorus acicularis,Pilophorus
cereolus,Pilophorus strumaticus,Cladia retipora,Stereocaulon paschale,Stereocaulon
taeniarum,Stereocaulon vesuvianum,Stereocaulon ramulosum,Lecidella
meiococca,Squamarina lentigera,Lecanora intumescens,Alectoria sarmentosa,Letharia
columbiana,Vulpicida pinastri,Cetraria slandica,Cornicularia normoerica,Pleurosticta
acetabulum,Xanthoparmelia conspersa,Evernia prunastri,Parmelia saxatilis,Hypogymnia physodes,Bunodophoron
scrobicu latum,Leifidium tenerum,Sphaerophorus globosus,Sphaerophorus
stereocauloides,Neophyllis melacarpa,Austropeltum glareosum,Lecania
cyrtella,Psora decipiens,Cliostomum griffithii,Toninia sedifolia,Bacidia
rosella,Caloplaca flavorubescens,Xanthoria elegans,Xanthoria parietina,Tephromela
atra,Thelomma mammosum,Texosporium sancti-jacobi,Cyphelium inquinans,Buellia
disciformis,Physcia aipolia,Cyphelium tigillare,Lecanora dispersa,Leprocaulon
sp.,Lecidea fuscoatra,Emericella nidulans,Aspergillus ustus,Aspergillus
versicolor,Aspergillus oryzae,Aspergillus parasiticus,Aspergillus
tamarii,Aspergillus flavus ,Aspergillus sojae,Aspergillus nomius,Aspergillus
avenaceus,Eurotium herbariorum,Eurotium rubrum,Aspergillus restrictus,Aspergillus
clavatus,Aspergillus fumigatus,Neosartorya fischeri,Aspergillus
terreus,Fennellia flavipes,Aspergillus wentii,Chaetosartorya cremea,Aspergillus
ochraceus,Aspergillus awamori,Aspergillus niger ,Aspergillus candidus,Aspergillus
cervinus,Penicillium allii,Penicillium expansum,Penicillium commune,Penicillium
freii,Penicilium notatum,Eupenicillium crustaceum,Geosmithia namyslowskii,Penicillium
namyslowskii,Eupenicillium javanicum,Chromocleista malachitea,Monascus
purpureus,Hamigera avellanea,Merimbla ingelheimense,Hamigera striata,Hemicarpenteles
ornatus,Penicilliopsis clavariiformis,Aspergillus zonatus,Aspergillus
sparsus,Chromocleista cinnabarina,Chromocleista cinnabarina,Talaromyces
flavus,Talaromyces bacillisporus,Talaromyces luteus,Talaromyces
luteus,Elaphomyces leveillei,Elaphomyces maculatus,Trichocoma paradoxa,Geosmithia
cylindrospora,Talaromyces eburneus,Talaromyces emersonii,Byssochlamys nivea,Thermoascus
crustaceus,Malbranchea dendritica,Malbranchea filamentosa,Auxarthron
zuffianum,Renispora flavissima,Malbranchea albolutea,Coccidioides
immitis ,Uncinocarpus reesii,Coccidioides immitis,Onygena equina,Pseudoamauroascus
australiensi,Castanedomyces australiensis,Trichophyton rubrum,Malbranchea
gypsea,Ascosphaera apis 2,Ascosphaera apis,Eremascus albus,Ajellomyces
capsulatus,Histoplasma capsulatum Eukaryo,Histoplasma capsulatum,Paracoccidioides
brasiliensis,Lacazia loboi,Lacazia loboi,Exophiala dermatidis,Graphium
calicioides,Capronia pilosella,Cladophialophora boppii,Cladophialophora
arxii,Fonsecaea pedrosoi,Cladophialophora devriesii,Phialophora
verrucosa,Capronia mansonii,Exophiala dermatitidis,Pullularia prototropha,Exophiala
dermatitidis,Sarcinomyces phaeomuriformis,Capronia coronata,Capronia
epimyces,Rhinocladiella phaeophora,Ramichloridium anceps,Capronia
fungicola,Capronia pilosella,Exophiala jeanselmei,Phaeococcomyces
exophialae,Phaeoannellomyces elegans,Nadsoniella nigra,Exophiala
sp.,Rhinocladiella atrovirens,Capronia dactylotricha,Capronia pulcherrima,Capronia
acutiseta,Cladophialophora sp.,Ceramothyrium linnaeae,Exophiala
mansonii,Phialophora sp.,Coniosporium perforans,Coniosporium apollinis,Coniosporium
per forans,Glyphium elatum,Euascomycetes sp.,Rhynchostoma minutum,Ceramothyrium
carniolicum,Caliciopsis pineam,Lasioderma serricorne yeast-like,Halosarpheia
marina,Halosarpheia viscosa,Phaeonectriella lignicola ,Halosarpheia
lotica,Halosarpheia retorquens,Nais inornata,Halosarpheia heteroguttulata,Halosarpheia
cincinnatula,Halosarpheia fibrosa,Halosarpheia spartinae ,Graphium
putredinis,Pseudallescheria boydii,Pseudallescheria ellipsoidea,Microascus
cirrosus,Graphium putredinis,Graphium tectonae,Graphium putredinis,Petriella
setifera,Lomentospora prolificans,Ceratocystis fimbriata,Graphium
penicillioides,Glomerella cingulata,Verticillium longisporum,Verticillium
dahliae,Volutella colletotrichoides,Plectosphaerella cucumerina,Cordyceps
yakusimensis,Cordyceps sobolifera,Cordyceps capitata,Cordyceps cochlidiicola,Cordyceps
prolifica,Cordyceps inegoensis,Tolypocladium inflatum,Cordyceps
paradoxa,Cordyceps japonica,Cordyceps heteropoda,Hamiltonaphis styraci
yeast-li,Cordyceps ramosopulvinata,Paecilomyces sp.,Cordyceps kanzashiana,Trichoderma
koningii,Trichoderma viride,Taphrina deformans,Hypocrea lutea,Hypomyces
chrysospermus,Volutella ciliata,Volutella ciliata,Chaetopsina fulva,Gibberella
pulicaris,Gibberella moniliformis,Botryotinia fuckeliana,Botryotinia
convoluta,Mycoarachis inversa,Cordyceps pruinosa,Cordyceps takaomontana,Botrytis
cinerea,Cordyceps brongniartii,Cordyceps militaris,Paecilomyces
tenuipes,Bionectria ochroleuca,Nectria ochroleuca,Nectria aureofulva,Nectria
cinnabarina,Geosmithia putterillii,Geosmithia lavendula,Lulworthia
fucicola,Chaetomium elatum,Neurospora crassa,Sordaria firmicola,Podospora
anserina,Ascovaginospora stellipala,Phyllachora graminis,Kionochaeta
ramifera,Kionochaeta spissa,Kionochaeta spissa,Kionochaeta ivoriensis,Meliola
juddiana,Meliola niessleana,Neurospora crassa,Ophioceras leptosporum,Magnaporthe
grisea,Pseudohalonectria falcata,Cryphonectria cubensis,Cryphonectria
havanensis,Endothia gyrosa,Leucostoma persoonii,Ophiostoma penicillatum,Pesotum
fragrans,Ophiostoma bicolor,Ophiostoma piceae,Ophiostoma floccosum,Ophiostoma
piliferum,Ophiostoma ulmi,Ophiostoma schenckii,Ophiostoma stenoceras,Ophiostoma
cucullatum,Ophiostoma europhioides,Ophiostoma ainoae,Papulosa amerospora,Ascolacicola
austriaca,Rhamphoria delicatula,sordariomycete sp.,Poronia punctata,Rosellinia
necatrix,Xylaria sp.,Xylaria polymorpha,Muscodor sp.,Barrmaelia
oxyacanthae,Hypoxylon fragiforme,Monographella nivalis,Pestalosphaeria
hansenii,Pestalosphaeria sp.,Hyponectria buxi,Monilinia fructicola,Monilinia
laxa,Sclerotinia sclerotiorum,dark septate endophyte DS16b,Euascomycetes
sp.,Neobulgaria premnophila,Blumeria graminis,Phyllactinia guttata,Spathularia
flavida,Cyttaria darwinii,Geomyces pannorum var. pannoru,Thelebolus
stercoreus,Ascozonus woolhopensis,Selenaspora guernisacii,Inermisia aggregata,Pyronema
domesticum,Otidea onotica,Glaziella aurantiaca,Cephaliophora tropica,Cephaliophora irregularis,Scutellospora
castanea,Geopyxis carbonaria Sarcoscypha austriaca,Pseudopithyella
minuscula,Kompsoscypha chudei Pithya cupressina,Nanoscypha tetraspora,Phillipsia
domingensis,Wynnea sp.,Cookeina tricholoma,Microstoma floccosum,Wolfina
aurantiopsis,Strobiloscypha keliae,Desmazierella acicola,Neournula
pouchetii,Chorioactis geaster,Donadinia sp.,Galiella rufa,Plectania
rhytidia,Sarcosoma mexicanum,Pseudoplectania nigrella,Tuber borchii
,Tuber gibbosum,Choiromyces meandriformis,Choiromyces venosus,Tuber
excavatum,Tuber magnatum, Tuber panniferum,Dingleya verrucosa,Reddellomyces
donkii,Labyrinthomyces varius,Helvella lacunosa,Wynnella silvicolor,Helvella
terrestris,Balsamia vulgaris,Barssia oregonensis,Balsamia magnata,
Underwoodia columnaris,Morchella elata 2,Morchella esculenta,Verpa
conica,Disciotis venosa,Fischerula subcaulis,Leucangium carthusianum,Verpa
bohemica,Pseudorhizina californica,Gyromitra esculenta 2,Gyromitra
melaleucoides,Discina macrospora,Gyromitra montana,Hydnotrya cerebriformis,Rhizina
undulata,Pachyphloeus citrinus,Cazia flexiascus,Terfezia arenaria,Peziza
echinospora,Terfezia terfezioides,Orbilia fimicola,Orbilia auricolor,Dactylella
oxyspora,Cephaliophora longispora,Cephaliophora muscicola,Arxiozyma
telluris,Saccharomyces transvaalensis,Saccharomyces dairensis,Saccharomyces
servazzii, Saccharomyces unisporus,Zygosaccharomyces bailii,Zygosaccharomyces
lentus,Zygosaccharomyces bisporus,Zygosaccharomyces mellis,Zygosaccharomyces
rouxii,Zygosaccharomyces rouxii 2,Torulaspora globosa,Zygosaccharomyces
florentinus,Candida colliculosa,Torulaspora delbrueckii,Torulaspora
pretoriensis,Zygosaccharomyces microellipso,Zygosaccharomyces mrakii,Torulaspora
delbrueckii,Saccharomyces kudriavzevii,Saccharomyces mikatae,Saccharomyces
cerevisiae ,Saccharomyces cariocanus,Saccharomyces bayanus,Saccharomyces
cerevisiae,Saccharomyces paradoxus,Saccharomyces pastorianus,Kluyveromyces polysporus,Kluyveromyces
yarrowii,Saccharomyces dairensis 2,Kluyveromyces africanus,Kluyveromyces
africanus,Saccharomyces rosinii,Kluyveromyces lodderae,Saccharomyces
spencerorum,Candida holmii,Saccharomyces barnettii,Saccharomyces
exiguus,Saccharomyces exiguus,Kazachstania viticola,Saccharomyces castellii,Candida
glabrata,,Kluyveromyces delphensis,Saccharomyces kluyveri,Eremothecium
gossypii,Holleya sinecauda,Kluyveromyces thermotolerans,Kluyveromyces
waltii,Zygosaccharomyces cidri,Zygosaccharomyces fermentati,Hanseniaspora
uvarum,Saccharomycodes ludwigii,Kluyveromyces marxianus,Kluyveromyces
wickerhamii,Kluyveromyces dobzhanskii,Kluyveromyces lactis Kluyveromyces
nonfermentati,Kluyveromyces aestuarii,Tetrapisispora nanseiensis,Tetrapisispora
arboricola,Kluyveromyces phaffii,Tetrapisispora iriomotensis,Kluyveromyces
blattae,Phaffomyces opuntiae,Phaffomyces opuntiae,Phaffomyces antillensis,Phaffomyces
thermotolerans,Candida insectalens,Candida silvatica,Pichia membranifaciens,Issatchenkia orientalis,Brettanomyces
anomalus 2,Dekkera anomala,Brettanomyces anomalus,Brettanomyces
bruxellensis,Dekkera bruxellensis,Dekkera custersiana,Dekkera naardenensis,Williopsis
salicorniae,Candida haemulonii,Candida tsuchiyae,Candida oregonensis,Candida
intermedia,Candida pseudointermedia,Candida intermedia,Candida akabanensis,Candida
melibiosica,Clavispora lusitaniae,Candida torresii,Candida agrestis,Metschnikowia
bicuspidata,Metschnikowia reukaufii,Metschnikowia pulcherrima,Candida
mogii,Williopsis californica,Williopsis pratensis,Starmera amethionina
var. pach,Starmera caribaea,Williopsis saturnus,Candida utilis,Pichia
anomala,Williopsis mucosa,Candida albicans,Candida dubliniensis,Candida
maltosa,Candida viswanathii,Candida lodderae,Candida tropicalis,Candida
sojae,Candida parapsilosis,Lodderomyces elongisporus,Candida fungicola,Candida
sagamina,Candida fukazawae,Candida mesenterica,Candida suecica,Candida
rugosa,Candida savonica,Candida tanzawaensis,Candida austromarina,Candida
sake,Candida kruisii,Candida insectamans,Candida lyxosophila,Candida
glaebosa,Candida pseudoglaebosa,Candida saitoana,Candida fluviatilis,Candida
palmioleophila,Candida palmioleophila,Candida shehatae var. insectos,Candida
shehatae var. ,shehatae,Candida shehatae var. lignosa,Candida coipomoensis,Candida
ergastensis,Candida chilensis,Candida glucosophila,Debaryomyces
hansenii,Debaryomyces castellii,Debaryomyces udenii,Candida psychrophila,Debaryomyces
hansenii var. fab,Candida fermenticarens,Candida oleophila,Candida zeylanoides,Candida
boleticola,Candida schatavii,Candida ralunensis,Candida krissii,Candida
laureliae,Candida santamariae var. membr,Candida santamariae var.
santa,Candida beechii,Candida sophiae-reginae,Candida natalensis,Candida
quercitrusa,Candida fragi,Candida xestobii,Candida fukuyamaensis,Taphrina
farlowii,Pichia guilliermondii,Pichia mexicana,Candida conglobata,Candida
insectorum,Candida aaseri,Candida butyri,Candida dendronema,Candida
naeodendra,Candida tenuis,Candida diddensiae,Candida friedrichii,Candida
membranifaciens,Candida buinensis,Candida atlantica,Candida atmosphaerica,Pichia
triangularis,Candida multigemmis,Pichia farinosa,Candida cylindracea,Endomyces
fibuliger,Saccharomycopsis fibuligera,Saccharomycopsis capsularis,Pachysolen
tannophilus,Endomyces geotrichum,Galactomyces geotrichum,Galactomyces
citri-aurantii,Galactomyces reessii„Dipodascus albidus„Dipodascus
albidus,Dipodascus geniculatus,Dipodascus australiensis,Dipodascus
armillariae,Dipodascus macrosporus,Geotrichum klebahnii,Dipodascus
aggrega tus,Geotrichum eriense,Geotrichum fermentans,Candida apicola,Candida
bombi,Starmerella bombicola,Candida geochares,Candida spandovensis,Zygoascus
hellenicus,Candida valdiviana,Arxula terrestris,Candida chiropterorum,Candida
drimydis,Dipodascopsis uninucleata,Waltomyces lipofer,Neolecta vitellina,Schizosaccharomyces
pombe,Schizosaccharomyces japonicus,Pneumocystis carinii,Pneumocystis
carinii,Taphrina mirabilis,Taphrina nana,Taphrina pruni,Taphrina
ulmi,Taphrina communis,Taphrina flavorubra,Taphrina deformans 2,Taphrina
prunisubcordatae,Taphrina populina,Taphrina wiesneri,Taphrina carnea,Taphrina
virginica,Taphrina letifera,Taphrina robinsoniana,Protomyces inouyei,Protomyces
lactucae,Protomyces macrosporus,Protomyces pachydermus,Saitoella
complicata,Leucoagaricus gongylophorus,Dictyonema pavonia,Tulostoma
macrocephala,Pluteus petasatus,Lentinula lateritia,Lentinula lateritia,Cyathus
striatus,Calvatia gigantea,Lycoperdon sp.Agaricus bisporus,Amanita
muscaria (fly agaric),Leucopaxillus albissimus,Tricholoma matsutake,Entoloma
strictius,Stropharia rugosoannulata Basidiomycete symbiont of Apte,Calathella
mangrovei,Nia vibrissa,Halocyphina villosa,Cyphellopsis anomala,Physalacria
maipoensis,Favolaschia intermedia subsp. ,Athelia bombacina,Cortinarius
iodes,Inocybe geophylla,Lepiota procera (parasol mushr,Limnoperdon
incarnatum,Panellus serotinus,Panellus stipticus,Hygrophorus sordidus,Humidicutis
marginata,Typhula phacorrhiza,Crucibulum laeve,Laccaria amethystina,Laccaria
pumila,Coprinus cinereus,Omphalina ericetorum,Pleurotus ostreatus,Fistulina
hepatica,Fistulina hepatica,Schizophyllum commune,Pleurotus tuberregium,Scleroderma
citrina,Xerocomus chrysenteron,Boletus satanas,Paxillus panuoides
Peniophora nuda,Peniophora nuda,Scytinostroma alutum,Echinodontium
tinctorium,Echinodontium tinctorium,Russula compacta,Russula compacta,Lentinellus
omphalodes,Lentinellus omphalodes,Lentinellus ursinus,Lentinellus ursinus,Clavicorona
pyxidata,Clavicorona pyxidata,Auriscalpium vulgare,Auriscalpium
vulgare,Hericium coralloides,Hericium erinaceum,Hericium ramosum,Hericium
ramosum,Stereum gausapatum,Stereum ostrea,Stereum hirsutum,Gloeocystidiellum
leucoxantha,Aleurodiscus botryosus,Stereum annosum,Bondarzewia berkeleyi,Albatrellus
skamanius,Heterobasidion annosum,Laxitextum bicolor,Trichaptum fusco-violaceum
Trichaptum biforme,TTrichaptum abietinu,Trichaptum abietinum,Trichaptum
laricinum,Basidioradulum radula,Hyphodontia alutaria,Inonotus hispidus,Phellinus
igniarius,Coltricia perennis,Schizopora paradoxa,Fomitopsis pinicola,Daedalea
quercina,Antrodia carbonica,Polyporus squamosus,Trametes suaveolens,Fomes
fomentarius,Lentinus tigrinus,Ganoderma australe,Dentocorticium
sulphurellum,Poria cocos,Cantharellus tubaeformis,Phaeolus schweinitzii,Laetiporus
sulphureus,Sparassis spathulata,Bjerkandera adusta,Oxyporus latemarginatus,Trichaptum
abietinum,Phlebia radiata,Phanerochaete chrysosporium,Rhizoctonia
zeae,Tretopileus sphaerophorus Ceriporia purpurea,Termitomyces cartilagineus,Panus
rudis,Steccherinum rhois,Meripilus giganteus,Albatrellus syringae,Spongipellis
unicolor,Hydnellum sp.,Thelephora sp.,Gloeophyllum sepiarium,Geastrum
saccatum,Sphaerobolus stellatus,Gomphus floccosus,Pseudocolus fusiformis,Ramaria
stricta,Clavariadelphus pistillaris,Multiclavula mucida,Clavulina
cristata,Hydnum repandum,Thanatephorus cucumeris,Thanatephorus praticola,Auricularia
auricula,Auricularia polytricha,Botryobasidium isabellinum,Botryobasidium subcoronatum,Oxyporus
sp.,Bullera grandispora,Mrakia frigida,Mrakia psychrophilia,Cystofilobasidium
capitatum,Leucosporidium larimarini,Tilletiopsis cremea,Tilletiopsis
lilacina,Tilletiopsis washingtonensis,Exobasidium vaccinii,Sympodiomycopsis
paphiopedili,Tilletiopsis pallescens,Tilletia caries,Tilletiaria
anomala,Tilletiopsis fulvescens,Tilletiopsis minor,Tilletiopsis
albescens,Graphiola cylindrica,Exobasidium rhododendri,Exobasidium
vaccinii,Dacrymyces chrysospermus,Dacrymyces stillatus,Calocera
cornea,Heterotextus alpinus,Trichosporon caseorum,Trichosporon lactis,Trichosporon
cutaneum,Asterotremella parasitica,Cryptococcus humicolus,Cryptococcus
albidus,Filobasidium floriforme,Holtermannia corniformis,Tremella
foliacea,Bullera schimicola,Bullera waltii,Bullera huiaensis,Bullera
boninensis,Bullera mrakii,Bullera coprosmaensis,Bullera oryzae,Bullera
derxii,Bullera sinensis,Bullera armeniaca,Dioszegia crocea,Bullera
variabilis,Filobasidiella neoformans,Tsuchiaea wingfieldii,Bullera
dendrophila,Bullera miyagiana,Bullera globospora,Tremella globospora,Kockovaella
imperatae,Kockovaella sacchari,Kockovaella thailandica,Kockovaella
phaffii,Kockovaella phaffii,Fellomyces fuzhouensis,Kockovaella machilophila,Fellomyces
distylii,Fellomyces ogasawarensis,Kockovaella schimae,Fellomyces
penicillatus,Fellomyces polyborus,Fellomyces horovitziae,Sterigmatosporidium
polymorphu,Tremella moriformis,Bullera hannae,Bullera penniseticola,Bullera
unica,Bulleromyces albus,Bullera pseudoalba,fungus sp.,Sirobasidium
magnum,Fibulobasidium inconspicuum,Taphrina california,Taphrina
maculans,Bensingtonia yamatoana,Sporobolomyces inositophilus,Sporobolomyces
singularis,Sporobolomyces falcatus,Sporobolomyces tsugae,Microbotryum
violaceum,Leucosporidium scottii,Bensingtonia intermedia,Sporidiobolus
pararoseus,Sporidiobolus pararoseus,Sporobolomyces roseus,Sporidiobolus
johnsonii,Sporidiobolus johnsonii,Sporidiobolus salmonicolor,Rhodotorula
glutinis,Rhodotorula mucilaginosa,Rhodosporidium toruloides,Rhodotorula
glutinis,Rhodosporidium fluviale,Sporidiobolus ruineniae,Mixia osmundae,Sporobolomyces
sasicola,Sporobolomyces xanthus,Kurtzmanomyces nectairei,Bensingtonia sp.,Sporobolomyces
ruber,Sterigmatomyces halophilus,Bensingtonia ingoldii,Bensingtonia
mu sae,Bensingtonia miscanthi,Bensingtonia subrosea,Kondoa malvinella,Bensingtonia
yuccicola,Bensingtonia phylladus,Bensingtonia ciliata,Bensingtonia
naganoensis,Sporobolomyces subbrunneus,Cronartium ribicola,Peridermium harknesseii,Hyalopsora
polypodii,Gymnoconia nitens,Nyssopsora echinata,Helicobasidium purpureum,Helicobasidium
corticioides,Helicogloea variabilis Sporobolomyces kluyveri-nielii,Sporobolomyces
phyllomatis,Sporobolomyces salicinus,Rhodotorula minuta,Sporobolomyces
foliicola,Sporobolomyces oryzicola,Sporobolomyces vermiculatus,Erythrobasidium
hasegawianum,Sporobolomyces yunnanensis,Sporobolomyces elongatus,Rhodotorula
lactosa,Archaeospora leptoticha,Paraglomus brasilianum,Glomus verruculosum,Glomus
intraradices,Glomus proliferum,Glomus etunicatum,Gigaspora margarita,Scutellospora
projecturata,Scutellospora castanea,Scutellospora cerradensis,Entrophospora
sp.,Glomus versiforme,Mucor circinelloides f. lusita,Mucor racemosus,Cepedea
virguloidea,Mucor ramosissimus,Ellisomyces anomalus,Backusella ctenidia,Circinomucor
circinelloides,Mucor racemosus,Opalina ranarum,Parasitella parasitica,Chaetocladium
brefeldii,Chaetocladium jonesii,Pilaira anomala,Thamnidium elegans,Helicostylum
elegans,Pirella circinans,Mucor mucedo,Mucor hiemalis f. hiemalis,Rhizomucor
variabilis,Zygorhynchus heterogamus,Actinomucor elegans,Dicranophora
fulva,Pilobolus umbonatus,Backusella circina,Mucor recurvus var.,indicus,Blakeslea
trispora,Poitrasia circinans,Choanephora cucurbitarum,Gilbertella
persicaria,Hyphomucor assamensis,Rhizopus microsporus var. micr,Rhizopus
microsporus var. rhiz,Rhizopus azygosporus,Rhizopus microsporus
var. chin,Rhizopus microsporus var. olig,Amylomyces rouxii,Rhizopus
arrhizus,Sporodiniella umbellata,Syzygites megalocarpus,Rhizopus
stolonifer,Benjaminiella poitrasii,Cokeromyces recurvatus,Mycotypha
africana,Mycotypha microspora,Kirkomyces cordense,Mucor amphibiorum,Mucor
indicus,Apophysomyces elegans,Saksenaea vasiformis,Radiomyces spectabilis,Phycomyces
blakesleeanus,Spinellus fusiger,Circinella umbellata,Phascolomyces
articulosus,Zychaea mexicana,Fennellomyces linderi,Thamnostylum
piriforme,Absidia corymbifera,Absidia blakesleeana,Dichotomocladium
elegans,Rhizomucor miehei,Rhizomucor pusillus,Thermomucor indicae-seudaticae,Protomycocladus
faisalabadensi,Absidia glauca,Chlamydoabsidia padenii,Absidia coerulea,Absidia
repens,Halteromyces radiatus,Cunninghamella bertholletiae,Cunninghamella
polymorpha,Cunninghamella elegans,Cunninghamella echinulata,Gongronella
butleri,Hesseltinella vesiculosa,Syncephalastrum monosporum var,Syncephalastrum
racemosum (U.),Mucor ramannianus,Blastocladiella emersonii,Entomophthora
muscae,Entomophthora schizophora,Eryniopsis ptycopterae,Entomophaga
aulicae,Pandora neoaphidis,Strongwellsea castrans,Zoophthora culisetae,Conidiobolus lamprauges,Conidiobolus
thromboides,Conidiobolus coronatus,Chytridiomycetes sp.,Smittium
simulii,Smittium commune,Smittium simulii,Smittium cylindrosporum,Smittium
imitatum,Smittium imitatum,Smittium tronadorium,Smittium orthocladii,Smittium
tipulidarum,Smittium dipterorum,Smittium megazygosporum,Smittium
cf. dipterorum,Smittium cf. phytotelmatum,Smittium phytotelmatum,Furculomyces
boomerangus,Smittium angustum,Furculomyces boomerangus,Smittium
morbosum,Smittium culicis,Smittium simulatum,Smittium culicis,Smittium
culicisoides,Smittium fecundum,Smittium annulatum,Smittium coloradense,Smittium
mucronatum,Smittium caudatum,Genistelloides hibernus,Capniomyces
stellatus,Smittium culisetae,Smittium culisetae,Smittium culisetae,Coemansia
reversa,Spirodactylon aureum,Coemansia braziliensis,Kickxella alabastrina,Linderina
pennispora,Dipsacomyces acuminosporus,Martensiomyces pterosporus,Spiromyces
aspiralis,Spiromyces minutus,Basidiobolus haptosporus,Basidiobolus
ranarum,Chytridium confervae,Neocallimastix sp.,Neocallimastix frontalis
2,Dissophora decumbens,Mortierella multidivaricata,Echinosporangium
transversale,Mortierella verticillata,Mortierella chlamydospora,Mortierella
wolfii,Mortierella polycephala,Monoblepharella elongata,Monoblepharella
mexicana,Monoblepharis hypogyna,Monoblepharis insignis,Hyaloraphidium
curvatum,Spizellomyces acuminatus,Filobasidiella neoformans 3,Candida
floricola.,Candida santjacobensis,dark septate,Metschnikowia agaves,Candida
etchellsii.,Pneumocystis carinii,Candida cantarellii.,Candida stellata.,Nadsoniella
nigra,Candida paludigena.,Candida versatilis.,Candida otoi,Candida
silvanorum.,Candida entomophila.,Candida sorbophila.,Candida uringiensis,Candida
antillancae.,Candida tepae.,Candida musae.,Candida ishiwadae.,Candida
ncudensis,Candida castrensis.,Candida vanderwaltii.,Metschnikowia
australis.,Candida fructus,Metschnikowia pulcherrima.,Candida bertae.,Wangiella
dermatitidis,Candida galacta.,Candida salmanticensis.,Candida parapsilosis,Candida
petrohuensis.,Candida populi.,Candida blankii.,Ochrolechia parella,Candida
rhagii.,Candida homilentoma.,Candida fennica.,Bulgaria inquinans,,Sporidiobolus
sp.,Wickerhamiella domercqiae.,Zygosaccharomyces rouxii,Metschnikowia
gruessii.,Metschnikowia reukaufii.,Metschnikowia bicuspidata.,Metschnikowia
krissii.,Candida anatomiae.,Arxula terrestris,Candida agrestis.,Glomerobolus
gelineus,Candida vinaria,Blastocladiella emersonii,Candida lactis-condensi,Metschnikowia
zobellii,Candida bondarzewiae,Candida sequanensis,Candida edax,Candida
ernobii,Candida karawaiewii,Saccharomyces cerevisiae,Barrmaelia
oxyacanthae,Arxula adeninivorans.Suitable cells for the above-mentioned methods are herbal, for example carrot roots, animal and human, for example Hela or HEK293, and in particular fungal cells. The processes can be carried out particularly well in cells of the following types:
Alternaria alternata, Clathrospora diplospora, Alternaria brassicae, Alternaria brassicicola, Alternaria raphani, Pleospora herbarum, Pyrenophora trichostoma Cochliobolus sativus, Cochliobolus heterostrophus, Setosphaeria monoceras Setosphaeria rostrata, Pleospora betae, Leptosphaeria maculans, Leptosphaeria microscopica, Ophiobolus herpotrichus, Cucurbitaria elongata, Phoma sp. Leptosphaeria Doliolum, Cucurbitaria Berberidis, Paraphaeosphaeria agavensis Paraphaeosphaeria nolinae, Paraphaeosphaeria obtusispora, Paraphaeosphaeria conglomerata, Paraphaeosphaeria quadriseptat, Paraphaeosphaeria filamentosa, Melanomma Sanguinarium, Paraphaeosphaeria glauco-punct, Septoria nodorum, Phoma herbarum Pleospora rudis, Helminthosporium solani, velutinum Helminthosporium, Leptosphaeria bicolor, Paraphaeosphaeria michotii, Paraphaeosphaeria pilleata, Cucurbidothis pityophila, Bimuria novae-zelandiae, Letendraea helminthicola, Kirschsteiniothelia elaterascu, Helicascus kanaloanus, Herpotrichia diffusa Mycosphaerella mycopappi, Herpotrichia juniperi, Byssothecium circinans, Lophiostoma crenatum, Trematosphaeria heterospora, Kirschsteiniothelia maritima, Sporomia lignicola, Westerdykella cylindrica, Westerdykella dispersa, Delichia didyma, Dendryphiopsis atra, Kirschsteiniothelia aethiops, Sarcinomyces petricola, Phaeosclera dematioides, Sarcinomyces crustaceus, Scytalidium dimidiatum, Scytalidium hyalinum, S cytalidium dimidiatum, Botryosphaeria ribis, Capnobotryella sp., bartschii Capnobotryella renispora, Coccodinium, Hortaea werneckii, Phaeotheca Fissurella, Acrospermum compressum, Acrospermum ramineum, Phaeotrichum benjaminii, Aureobasidium pullulans, Discosphaerina fagi, dothidea hippophaeos, dothidea insculpta, Stylodothis puccinioides, dothidea ribesia, Delphi Ella strobiligena, Myriangium duriaei, Myriangium duriaei, Piedraia hortae, Raciborskiomyces longisetosum, Capnodium citri, Euascomycetes sp., Thecotheus holmskjoldii, Lasallia rossica, Baeomyces rufus, Anamylopsora pulcherrima, Placopsis gelida, Trapelia involuta, Trapelia placodioides, Pertusaria trachytallina, Ochrolechia parella, Coccotrema cucurbitula , Lepolichen coccophorus, Diploschistes rampoddensis, Diploschistes thunbergianus, Graphis scripta, Conotrema populorum, Gyalecta ulmi, Dibaeis, Stenocybe pullatula, Sphinctrina turbinata, Cladonia sulphuri na, Cladonia bellidiflora, Cladonia subcervicornis, Metus conglomeratus, Pilophorus robustus, Pilophorus acicularis, Pilophorus cereolus, Pilophorus strumaticus, Cladia retipora, Stereocaulon paschale, Stereocaulon taeniarum, Stereocaulon vesuvianum, Stereocaulon ramulosum, Lecidella meiococca, Squamarina lentigera, Lecanora intumescens, Alectoria sarmentosa, Letharia columbiana, Vulpicida pinastri, Cetraria slandica, Cornicularia normoerica, Pleurosticta acetabulum, Xanthoparmelia conspersa, Evernia prunastri, Parmelia saxatilis, Hypogymnia physodes, Bunodophoron scrobicu latum, Leifidium tenerum, Sphaerophorus globosus, Sphaerophorus stereocauloides, Neophyllis melacarpa, Exogenous glareosum, Lecania cyrtella, Psora decipiens, Cliostomum griffithii, Toninia sedifolia, Bacidia rosella, Caloplaca flavorubescens, Xanthoria elegans, Xanthoria parietina, Tephromela atra, Thelomma mammosum, Texosporium sancti-jacobi, Cyphelium inquinans, Buellia disciformis, Physcia aipolia, Cyphelium tigillare, Lecanora dispersa, Leprocaulon sp. Lec Aspergillus parasiticus, Aspergillus tamarii, Aspergillus flavus, Aspergillus sojae, Aspergillus nomius, Aspergillus avenaceus, Eurotium herbariorum, Eurotium rubrum, Aspergillus restrictus, Aspergillus clavatus, Aspergillus fumigatus, Neosartorya fischeri, Aspergillus versicolor, Aspergillus oryzae, Aspergillus parasiticus , Aspergillus terreus, Fennellia flavipes, Aspergillus wentii, Chaetosartorya cremea, Aspergillus ochraceus, Aspergillus awamori, Aspergillus niger, Aspergillus candidus, Aspergillus cervinus, Penicillium allii, Penicillium expansum, Penicillium commune, Penicillium freii, Penicillium notatum, Eupenicillium crustaceum, Geosmithia namyslowskii, Penicillium namyslowskii, Eupenicillium javanicum, Chromocleista malachitea, Monascus purpureus, Hamigera avellanea, Merimbla ingelheimense, Hamigera striata, Hemicarpenteles ornatus, Penicilliopsis clavariiformis, Aspergillus zonatus, Aspergillus sparsus, Chromocleista cinnabarina, Chromocleista cinnabarina, Talaromyces flavus, Talaromyces b acillisporus, Talaromyces luteus, Talaromyces luteus, Elaphomyces leveillei, Elaphomyces maculatus, Trichocoma paradoxa, Geosmithia cylindrospora, Talaromyces eburneus, Talaromyces emersonii, Byssochlamys nivea, Thermoascus crustaceus, Malbranchea dendritica, Malbranchea filamentosa, Auxarthron zuffianum, Renispora flavissima, Malbranchea albolutea, Coccidioides immitis, Uncinocarpus reesii, Coccidioides immitis, Onygena equina, Pseudoamauroascus australiensi, Castanedomyces australiensis, Trichophyton rubrum, Malbranchea gypsea, Ascosphaera apis 2, Ascosphaera apis, Eremascus albus, Ajellomyces capsulatus, Histoplasma capsulatum Eukaryo, Histoplasma capsulatum, Paracoccidioides brasiliensis, Lacazia loboi, Lacazia loboi, Exophiala dermatidis, Graphium calicioides, Capronia pilosella, Cladophialophora boppii, Cladophialophora arxii, Fonsecaea pedrosoi, Cladophialophora devriesii, Phialophora verrucosa, Capronia mansonii, Exophiala dermatitidis, Pullularia prototropha, Exophiala dermatitidis, Sarcinomyces phaeomuriformis, Capronia coronata, Capronia epimyces, Rhinocladiella phaeophora, Ramichloridium anceps, Capronia fungicola, Capronia pilosella, Exophiala jeanselmei, Phaeococcomyces exophialae, Phaeoannellomyces elegans, Nadsoniella nigra, Exophiala sp., Rhinocladiella atrovirens, Capronia dactylotricha, Capronia pulcherrima, Capronia acutiseta, Cladophialophora sp., Ceramothyrium linnaeae, Exophiala mansonii, Phialophora sp., Coniosporium perforans, Coniosporium apollinis, Coniosporium per forans, Glyphium elatum, Euascomycetes sp., Rhynchostoma minutum, Ceramothyrium carniolicum, Caliciopsis pineam, Lasioderma serricorne yeast-like, Halosarpheia marina, Halosarpheia viscosa, Phaeonectriella lignicola, Halosarpheia lotica, Halosarpheia retorquens, Nais inornata, Halosarpheia heteroguttulata, Halosarpheia cincinnatula, Halosarpheia fibrosa, Halosarpheia spartinae, Graphium putredinis, Pseudallescheria boydii, Pseudallescheria ellipsoidea, Microascus cirrosus, Graphium putredinis, Graphium tectonae, Graphium putredinis, Petriella setifera, Lomentospora prolificans, Ceratocystis fimbriata, Graphium penicillioides, Glomerella cingulata, Verticillium longisporum, dahliae Verticillium, Volutella colletotrichoides, Plectosphaerella cucumerina, Cordyceps yakusimensis, Cordyceps sobolifera, Cordyceps capitata, Cordyceps cochlidiicola, Cordyceps prolifica, Cordyceps inegoensis, inflatum Tolypocladium, Cordyceps paradoxa, Cordyceps japonica, Cordyceps heteropoda, Hamiltonaphis styraci yeast-li, Cordyceps ramosopulvinata, Paecilomyces sp., Cordyceps kanzashiana, Trichoderma koningii, Trichoderma viride, Taphrina deformans, Hypocrea lutea, Hypomyces chrysosperm, Volutella ciliata, Volutella ciliata, Chaetopsina fulva, Gibberella pulicaris, Gibberella moniliformis, Botryotinia Fattyelia, Botryotinia convoluta, Mycoarachis inversa, Cordyceps pruinosa, Cordyceps takaomontana, Botrytis cinerea, Cordyceps brongniartii, Cordyceps militaris, Paecilomyces tenuipes, Bionectria ochroleuca, Nectria ochroleuca, Nectria aureofulva, Nectria cinnabarina, Geosmithia putterillii, Geosmithia lavendula, Lu lworthia fucicola, Chaetomium elatum, Neurospora crassa, Sordaria firmicola, Podospora anserina, Ascovaginospora stellipala, Phyllachora graminis, Kionochaeta ramifera, Kionochaeta spissa, Kionochaeta spissa, Kionochaeta ivoriensis, Meliola juddiana, Meliola niessleana, Neurospora crassa, Ophioceras leptosporum, Magnaporthe grisea, Pseudohalonectria falcata , Cryphonectria cubensis, Cryphonectria havanensis, Endothia gyrosa, leucostoma persoonii, Ophiostoma penicillatum, Pesotum fragrans, Ophiostoma bicolor, piceae Ophiostoma, Ophiostoma floccosum, schenckii Ophiostoma piliferum, Ophiostoma ulmi, Ophiostoma, Ophiostoma stenoceras, Ophiostoma cucullatum, Ophiostoma ainoae europhioides, Ophiostoma, papulosa amerospora, Ascolacicola austriaca, Rhamphoria delicatula, sordariomycete sp., Poronia punctata, Rosellinia necatrix, Xylaria sp., Xylaria polymorpha, Muscodor sp., Barrmaelia oxyacanthae, Hypoxylon fragiforme, Monographella nivalis, Pestalosphaeria hansenii, Pestalosphaeria sp., Hyponectria buxi, Monilinia fructicola , Monilinia laxa , Sclerotinia sclerotiorum, dark septate endophyte DS16b, Euascomycetes sp., Neobulgaria premnophila, Blumeria graminis, Phyllactinia guttata, Spathularia flavida, Cyttaria darwinii, Geomyces pannorum var. pannoru, woolhopensis Thelebolus stercoreus, Ascozonus, Selenaspora guernisacii, Inermisia aggregata, Pyronema domesticum, Otidea onotica, Glaziella aurantiaca, Cephaliophora tropica, Cephaliophora irregularis, Scutellospora castanea, Geopyxis carbonaria Sarcoscypha austriaca, Pseudopithyella minuscula, Kompsoscypha chudei Pithya cupressina, Nanoscypha tetraspora , Phillipsia domingensis, Wynnea sp., Cookeina tricholoma, Microstoma floccosum, Wolfina aurantiopsis, Strobiloscypha keliae, Desmazierella acicola, Neournula pouchetii, Chorioactis geaster, Donadinia sp., Galiella rufa, Plectania rhytidia, Sarcosoma mexicanum, Pseudoplectania nigrella, Tuber borchii, Tuber gibbosum , Choiromyces meandriformis, Choiromyces venosus, Tuber excavatum, Tuber magnatum, Tuber panniferum, Dingleya verrucosa, Reddellomyces donkii, Labyrinthomyces varius, Helvella lacunosa, Wynnella silvicolor, Helvella terrestris, Balsamia vulgaris, Barssia oregonensis, Balsamia magnata, Underwoodia columnaris, Morchella elata 2, Morchella esculenta Verpa conica, Disciotis venosa, Fischerula subcaulis, Leucangium carthusianum, Verpa bohemica, Pseudorhizina californica, Gyromitra esculenta 2, Gyromitra melaleucoides, Discina macrospora, Gyromitra montana, Hydnotrya cerebriformis, Rhizina undulata, Pachyphloeus citrinus, Cazia flexiascus, Terfezia arenaria, Peziza echinospora, Terfezia terfezioides, Orbilia fimicola, Orbilia auricolor, Dactylella oxyspora, Cephaliophora longispora, Cephaliophora muscicola, Arxiozyma Telluris, Saccharomyces transvaalensis, Saccharomyces dairensis, Saccharomyces servazzii, Saccharomyces unisporus, Zygosaccharomyces bailii, Zygosaccharomyces lentus, Zygosaccharomyces bisporus, Zygosaccharomyces mellis, Zygosaccharomyces rouxii, Zygosaccharomyces rouxii 2, Torulaspora globosa, Zygosaccharomyces florentinus, Candida colliculosa, Torulaspora delbrueckii, Torulaspora pretoriensis, Zygosaccharomyces microellipso, Zygosaccharomyces mrakii, Torulaspora delbrueckii, Saccharomyces kudriavcevii, Saccharomyces mikatae, Saccharomyces cerevisiae, Saccharo myces cariocanus, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces pastorianus, Kluyveromyces polysporus, Kluyveromyces yarrowii, Saccharomyces dairensis 2, Kluyveromyces africanus, Kluyveromyces africanus, Saccharomyces rosinii, Kluyveromyces lodderae, Saccharomyces spencerorum, Candida holmii, Saccharomyces barnettii, Saccharomyces exiguus, Saccharomyces exiguus, Kazachstania viticola, Saccharomyces castellii, Candida glabrata gossypii ,, Kluyveromyces delphensis, Saccharomyces kluyveri, Eremothecium, Holleya sinecauda, Kluyveromyces thermotolerans, Kluyveromyces waltii, Zygosaccharomyces cidri, Zygosaccharomyces fermentati, Hanseniaspora uvarum, Saccharomycodes ludwigii, Kluyveromyces marxianus, Kluyveromyces wickerhamii, Kluyveromyces dobzhanskii Kluyveromyces lactis Kluyveromyces nonfermentati, Kluyveromyces aestuarii, Tetrapisispora nanseiensis, Tetrapisispora arboricola, Kluyveromyces phaffii, Tetrapisispora iriomotensis, Kluyveromyces blattae, Phaffomyces opuntiae, Phaffomyces op untiae, Phaffomyces antillensis, Phaffomyces thermotolerans, Candida insectalens, Candida silvatica, Pichia membranifaciens, Issatchenkia orientalis, Brettanomyces anomaly 2, Dekkera anomala, Brettanomyces anomalis, Brettanomyces bruxellensis, Dekkera bruxellensis, Dekkera custersiana, Dekkera naardenensis, Williopsis salicorniae, Candida haemulonii, Candida tsuchiyae , Candida oregonensis, Candida intermedia, Candida pseudointermedia, Candida intermedia, Candida akabanensis, Candida melibiosica, Clavispora lusitaniae, Candida torresii, Candida agrestis, Metschnikowia bicuspidata, Metschnikowia reuchnii, Metschnikowia pulcherrima, Candida mogii, Williopsis californica, Williopsis pratensis, Starmera amethionina var. Candida utilopis, Pichia anomala, Williopsis mucosa, Candida albicans, Candida dubliniensis, Candida maltosa, Candida viswanathii, Candida iodidea, Candida tropicalis, Candida sojae, Candida parapsilosis, Lodderomyces elongisporus, Candida fungicola, Candida sagamina, Candida candida mesenterica, Candida suecica, Candida rugosa, Candida savonica, Candida tanzawaensis, Candida austromarina, Candida sake, Candida kruisii, Candida insectamans, Candida lyxosophila, Candida glaebosa, Candida pseudoglaebosa, Candida saitoana, Candida fluviatilis, Candida palmioleophila, Candida palmioleophila, Candida shehatae var. Insectos, Candida shehatae var., Shehatae, Candida shehatae var. Lignosa, Candida coipomoensis, Candida ergastensis, Candida chilensis, Candida glucosophila, Debaryomyces hansenii, Debaryomyces castellii, Debaryomyces udenii, Candida psychrophila, Debaryomyces hansenii var. Fab, Candida fermenticarens, Candida oleophila, Candida zeylanoides, Candida boleticola, Candida schatavii, Candida ralunensis, Candida krissii, Candida laureliae, Candida santamariae var. membr, Candida santamariae var. santa, Candida beechii, Candida sophiae-reginae, Candida natalensis, Candida quercitrusa, Candida fragi, Candida xestobii, Candida fukuyamaensis, Taphrina farlowii, Pichia guilliermondii, Pichia mexica Candida conglobata, Candida insectorum, Candida aaseri, Candida butyri, Candida dendronema, Candida naeodendra, Candida tenuis, Candida diddensiae, Candida friedrichii, Candida membranifaciens, Candida buinensis, Candida atlantica, Candida atmospheraerica, Pichia triangularis, Candida multigemmis, Pichia farinosa, Candida cylindracea, Endomyces fibuliger, Saccharomycopsis fibuligera, Saccharomycopsis capsularis, Pachysolen tannophilus, Endomyces geotrichum, Galactomyces geotrichum, Galactomyces citri-aurantii, Galactomyces reessii "Dipodascus albidus" Dipodascus albidus, Dipodascus geniculatus, Dipodascus australiensis, Dipodascus armillariae, Dipodascus macrosporus, Geotrichum klebahnii, Dipodascus aggrega tus, Geotrichum eriense, Geotrichum fermentans, Candida apicola, Candida bombi, Starmerella bombicola, Candida geochares, Candida spandovensis, Zygoascus hellenicus, Candida valdiviana, Arxula terrestris, Candida chiropterorum, Candida drimydis, Dipodascopsis uninucleata, Waltomyces lipofer, Neolecta vitellina, Schizosacchar omyces pombe, Schizosaccharomyces japonicus, Pneumocystis carinii, Pneumocys Tis carinii, Taphrina mirabilis, Taphrina nana, Taphrina pruni, Taphrina ulmi, Taphrina communis, Taphrina flavorubra, Taphrina deformans 2, Taphrina prunisubcordatae, Taphrina populina, Taphrina wiesneri, Taphrina carnea, Taphrina virginica, Taphrina letifera, Taphrina robinsoniana, Protomyces inouyei, Protomyces lactucae, Protomyces macrosporus, Protomyces pachyderm, Saitoella complicata, Leucoagaricus gongylophorus, Dictyonema pavonia, Tulostoma macrocephala, Pluteus petasatus, Lentinula lateritia, Lentinula lateritia, Cyathus striatus, Calvatia gigantea, Lycoperdon sp.Agaricus bisporus, Amanita muscaria (fly agaric), Leucopaxillus albissimus , Tricholoma matsutake, Entoloma strictius, Stropharia rugosoannulata Basidiomycete symbiont of Apte, Calathella mangrovei, Nia vibrissa, Halocyphina villosa, Cyphellopsis anomala, Physalacria maipoensis, Favolaschia intermedia subsp. , Athelia bombacina, Cortinarius iodes, Inocybe geophylla, Lepiota procera (parasolus, Limnoperdon incarnatum, Panellus serotinus, Panellus stipticus, Hygrophorus sordidus, Humidicutis marginata, Typhula phacorrhiza, Crucibulum laeve, Laccaria amethystina, Laccaria pumila, Coprinus cinereus, Omphalina ericetorum, Pleurotus ostreatus, Fistulina hepatica, Fistulina hepatica, Schizophyllum commune, Pleurotus tuberregium, Scleroderma citrina, Xerocomus chrysenteron, Boletus satanas, Paxillus panuoides Peniophora nuda, Peniophora nuda, Scytinostroma alutum, Echinodontium tinctorium, Echinodontium tinctorium, Russula compacta, Russula compacta, Lentinellus omphalodes, Lentinellus omphalodes, Lentinellus ursinus, Lentinellus ursinus, Clavicorona pyxidata, Clavicorona pyxidata, Auriscalpium vulgare, Auriscalpium vulgare, Hericium coralloides, Hericium erinaceum, Hericium ramosum, Hericium ramosum, Stereum gausapatum, Stereum ostrea, Stereum hirsutum, Gloeocystidiellum leucoxantha, Aleurodiscus botryosus, Stereum annosum, Bondarzewia berk eleyi, Albatrellus skamanius, Heterobasidion annosum, Laxitextum bicolor, Trichaptum fusco-violaceum Trichaptum biforme, TTrichaptum abietinu, Trichaptum abietinum, Trichaptum laricinum, Basidioradulum radula, Hyphodontia alutaria, Inonotus hispidus, Phellinus igniarius, Coltricia perennis, Schizopora paradoxa, Fomitopsis pinicola, Daedalea quercina , Antrodia carbonica, Polyporus squamosus, Trametes suaveolens, Fomes fomentarius, Lentinus tigrinus, Ganoderma australe, Dentocorticium sulphurellum, Poria cocos, Cantharellus tubaeformis, Phaeolus schweinitzii, Laetiporus sulphureus, Sparassis spathulata, Bjerkandera adusta, Oxyporus latemarginatus, Trichaptum abietinum, Phlebia radiata, Phanerochaete chrysosporium, Rhizoctonia zeae, Tretopileus sphaerophorus Ceriporia purpurea, Termitomyces cartilagineus, Panus rudis, Steccherinum rhois, Meripilus giganteus, Albatrellus syringae, Spongipellis unicolor, Hydnellum sp., Thelephora sp., Gloeophyllum sepiarium, Geastrum saccatum, Sphaerobolus stellatus, Gomphus floccosus, Pseudocolus fusif ormis, Ramaria stricta, Clavariadelphus pistillaris, Multiclavula mucida, Clavulina cristata, Hydnum repandum, Thanatephorus cucumeris, Thanatephorus praticola, Auricularia auricula, Auricularia polytricha, Botryobasidium isabellinum, Botryobasidium subcoronatum, Oxyporus sp., Bullera grandispora, Mrakia frigida, Mrakia psychrophilia, Cystofilobasidium capitatum , Leucosporidium larimarini, Tilletiopsis cremea, Tilletiopsis lilacina, Tilletiopsis washingtonensis, vaccinii Exobasidium, Sympodiomycopsis paphiopedili, Tilletiopsis pallescens, Tilletia caries, Tilletiaria anomala, Tilletiopsis fulvescens, Tilletiopsis minor, Tilletiopsis albescens, rhododendri Graphiola cylindrica, Exobasidium, vaccinii Exobasidium, Dacrymyces chrysospermus, Dacrymyces Stillatus, Calocera cornea, Heterotextus alpinus, Trichosporon caseorum, Trichosporon lactis, Trichosporon cutaneum, Asterotremella parasitica, Cryptococcus humicolus, Cryptococcus albidus, Filobasidium floriforme, Holtermannia corniformis, Tremella foliacea, Bullera schimicola, Bullera walsii, Bullera huiaensis, Bullera boninensis, Bullera mrakii, Bullera coprosmaensis, Bullera oryzae, Bullera derxii, Bullera sinensis, Bullera armeniaca, Dioszegia crocea, Bullera variabilis, Filobasidiella neoformans, Tsuchiaea wingfieldii, Bullera dendrophila, Bullera miyagiana, Bullera globospora, Tremella globospora, Kockovaella imperatae, Kockovaella sacchari, Kockovaella thailandica, Kockovaella phaffii, Kockovaella phaffii, Fellomyces fuzhouensis, Kockovaella machilophila, Fellomyces distylii, Fellomyces ogasawarensis, Kockovaella schimae, Fellomyces penicillatus, Fellomyces polyborus, Fellomyces horovitziae, Sterigmatosporidium polymorphu, Tremella moriformis, Bullera hannae, Bullera penniseticola Bullera unica, Bulleromyces albus, Bullera pseudoalba, fungus sp., Sirobasidium magnum, Fibulobasidium inconspicuum, Taphrina california, Taphrina maculans, Bensingtonia yamatoana, Sporobolomyces inositophilus, Sporobolomyces singularis, Sporobolomyces falcatus, Sporobolomyces tsugae, Microbotryum violaceum, Leucosporidium scotti i, Bensingtonia intermedia, Sporidiobolus pararoseus, Sporidiobolus pararoseus, Sporobolomyces roseus, Sporidiobolus johnsonii, johnsonii Sporidiobolus, salmonicolor Sporidiobolus, Rhodotorula glutinis, Rhodotorula mucilaginosa, Rhodosporidium toruloides, Rhodotorula glutinis, Rhodosporidium fluviale, ruineniae Sporidiobolus, mixiomycetes, Sporobolomyces sasicola, Sporobolomyces xanthus, Kurtzmanomyces nectairei, Bensingtonia sp., Sporobolomyces ruber, Sterigmatomyces halophilus, Bensingtonia ingoldii, Bensingtonia sae, Bensingtonia miscanthi, Bensingtonia subrosea, Kondoa malvinella, Bensingtonia yuccicola, Bensingtonia phylladus, Bensingtonia ciliata, Bensingtonia naganoensis, Sporobolomyces subbrunneus, Cronartium ribicola, Peridermium harknesseii, Hyalopsora polypodii, Gymnoconia nitens, Nyssopsora echinata, Helicobasidium purpureum, Helicobasidium corticioides, Helicogloea variabilis Sporobolomyces kluyveri-nielii, Sporobolomyces phyllomatis, Sporobolomyces salicinus, Rhodotorula minuta, Sporobolomyces foliicola, Spor obolomyces oryzicola, Sporobolomyces vermiculatus, Erythrobasidium hasegawianum, Sporobolomyces yunnanensis, Sporobolomyces elongatus, Rhodotorula lactosa, Archaeospora leptoticha, Paraglus brasilianum, Glomus verruculosum, Glomus intraradices, Glomus proliferum, Glomus etunicatum, Gigaspora margarita, Scutellospora projecturata, Scu tellospora castanea, Scutello spora cerradensis, Entrophospora sp., Glomus versiforme, Mucor circinelloides f. lusita, Mucor racemosus, Cepedea virguloidea, Mucor ramosissimus, Ellisomyces anomalus, Backusella ctenidia, Circinomucor circinelloides, Mucor racemosus, Opalina ranarum, Parasitella parasitica, Chaetocladium brefeldii, Chaetocladium jonesii, Pilaira anomala, Thamnidium elegans, Helicostylum elegans, Pirella circinans, Mucor mucedo, Mucor hiemalis f. hiemalis, Rhizomucor variabilis, Zygorhynchus heterogamus, Actinomucor elegans, Dicranophora fulva, Pilobolus umbonatus, Backusella circina, Mucor recurvus var., indicus, Blakeslea trispora, Poitrasia circinans, Choanephora cucurbitarum, Gilbertella persicaria, Hyphomucor assamensis, Rhizopus microsporus var. micr, Rhizopus microsporus rhiz, Rhizopus azygosporus, Rhizopus microsporus var. chin, Rhizopus microsporus var. olig, Amylomyces rouxii, Rhizopus arrhizus, Sporodiniella umbellata, Syzygites megalocarpus, Rhizopus stolonifer, Benjaminiella poitrasii, Cokeromyces recurvatus, Mycotypha africana, Mycotypha microspora, Kirkomyces cordense, Mucor amphibiorum, Mucor indicus, Apophysomyces elegans, Saksenaea vasiformis, Radiomyces spectabilis, Phycomyces blakesleeanus, Spinellus fusiger, Circinella umbellata, Phascolomyces articulosus, Zychaea mexicana, Fennellomyces linderi, Thamnostylum piriforme, Absidia corymbifera, Absidia blakesleeana, Dichotomocladium elegans, Rhizomucor miehei, Rhizomucor pusillus, Thermomucor indicae seudaticae, Protomycocladus faisalabadensi, Absidia glauca, Chlamydoabsidia padenii, Absidia coerulea, Absidia repens, Halteromyces radiatus, Cunninghamella bertholletiae, Cunninghamella polymorpha, Cunninghamella elegans, Cunninghamella echinulata, Gongronella butleri, Hesseltinella vesiculosa, Syncephalastrum monosporum var, Syncephalastrum racemosum (U.), Mucor ramannianus, Blastocladiella emersonii, Entomophthora muscae, Entomophthora Schizophora, Eryniopsis ptycopterae, aulicae Entomophaga, Pandora neoaphidis, Strongwellsea castrans, Zoophthora culisetae, Conidiobolus lamprauges, Conidiobolus thromboides, Conidiobolus coronatus, sp Chytridiomycetes., simulii Smittium, simulii Smittium commune, Smittium, Smittium cylindrosporum , Immittatum, Immitatum, Sodium tronadorium, Smittium orthocladii, Sutrum tipulidarum, Sodium dipterorum, Sodium megazygosporum, Sodium cf. dipterorum, Smittium cf. phytotelmatum, Smittium phytotelmatum, Furculomyces boomerangus, Sardinian angustum, Furculomyces boomerangus, Smit tium morbosum, culinarum, simulatum, culinarum, culinarisoidum, fecundum, annulatum, smittium coloradense, smittium mucronatum, caudate, genistelloides hibernus, capniomyces stellatus, culisetae, culisetae, culisetae, coemansia reversa, spirodactylon aureum , Coemansia braziliensis, Kickxella alabastrina, Linderina pennispora, Dipsacomyces acuminosporus, Martensiomyces pterosporus, Spiromyces aspiralis, Spiromyces minutus, Basidiobolus haptosporus, Basidiobolus ranarum, Chytridium confervae, sp Neocallimastix., Neocallimastix frontalis 2, Dissophora decumbens, Mortierella multidivaricata, transverse Echinosporangium, Mortierella verticillata , Mortierella chlamydospora, Mortierella wolfii, Mortierella polycephala, Monoblepharella elongata, Monoblepharella mexicana, Monoblepharis hypogyna, Monoblepharis insignis, Hyaloraphidium curvatum, Spizellomyces acuminatus, Filobasidiella neoformans 3, Candida floricola., Candida santjacobensis, dark septate , Metschnikowia agaves, Candida etchellsii., Pneumocystis carinii, Candida cantarellii., Candida stellata., Nadsoniella nigra, Candida paludigena., Candida versatilis., Candida otoi, Candida silvanorum., Candida entomophila., Candida sorbophila., Candida uringiensis, Candida antillancae , Candida tepae., Candida musae., Candida ishiwadae., Candida ncudensis, Candida castrensis., Candida vanderwaltii., Metschnikowia australis., Candida fructus, Metschnikowia pulcherrima., Candida bertae., Wangiella dermatitidis, Candida galacta., Candida salmanticensis. , Candida parapsilosis, Candida petrohuensis., Candida populi., Candida blankii., Ochrolechia parella, Candida rhagii., Candida homilentoma., Candida fennica., Bulgaria inquinans, Sporidiobolus sp., Wickerhamiella domercqiae, Zygosaccharomyces rouxii, Metschnikowia gruessii. Metschnikowia reukaufii., Metschnikowia bicuspidata., Metschnikowia krissii., Candida anatomiae, Arxula terrestris, Candida agrestis, Glomerobolus gelineus, Candida vinaria, Blastocladiella emersonii, Candida la ctis-condensi, Metschnikowia zobellii, Candida bondarzewiae, Candida sequanensis, Candida edax, Candida ernobii, Candida karawaiewii, Saccharomyces cerevisiae, Barrmaelia oxyacanthae, Arxula adeninivorans.
Besonders geeignete Ausführungsformen der erfindungsgemässen Verfahren sind dadurch gekennzeichnet, dass die DNA-II-Sequenz eine DNA-Sequenz ist, die für ein Polypeptid gemäss SEQ ID NO: 2,4,6 oder 8 codiert oder eine DNA-Sequenz ist, die für ein Polypeptid codiert, das 80% Identität zu SEQ ID NO: 2,4,6 oder 8 besitzt, umfasst.Especially suitable embodiments the inventive Methods are characterized in that the DNA II sequence is a DNA sequence is responsible for a polypeptide according to SEQ ID NO: 2,4,6 or 8 encodes or is a DNA sequence encoding a polypeptide coded, 80% identity to SEQ ID NO: 2,4,6 or 8 comprises.
Besonders bevorzugt sind solche DNA-II-Sequenzen, die für ein Polypeptid mit Laccaseaktivität codieren, das 85, 90, 92, 94, 95, 96, 97, 98, 99 % Identität zu SEQ ID NO: 2,4,6 oder 8 besitzt. Die Identität zwischen zwei Polypeptidsequenzen wird dabei anhand der GCG Software, die von der University of Wisconsin entwickelt worden ist, mittels des Programms GAP unter Verwendung der vom Programm als Standardwerte vorgeschlagenen Einstellungen für gap penalty, ermittelt.Especially preferred are those DNA II sequences which code for a polypeptide with laccase activity, the 85, 90, 92, 94, 95, 96, 97, 98, 99% identity to SEQ ID NO: 2,4,6 or 8 owns. The identity between two polypeptide sequences is determined using the GCG software, which has been developed by the University of Wisconsin, by means of of the GAP program using the program's default values Settings for gap penalty, determined.
Als Laccase-Aktivität wird die Fähigkeit bezeichnet, phenolische Substanzen zu oxidieren wie sie beispielsweise in Machuca et al., 1998, auf Seite 218 (Determination of laccase type PO activity) beschrieben wird.Laccase activity is the ability to oxidize phenolic substances as described, for example, in Machuca et al., 1998, at page 218 (Determination of laccase type PO activity) becomes.
Das Einführen von DNA-Molekülen in Zellen geschieht durch dem Fachmann bekannte geläufige Klonierungs- und Transfektionsmethoden, wie beispielsweise Co-Präzipitation, Protoplastenfusion, Elektroporation, retrovirale Transfektion und dergleichen, verwendet, um die genannten Nukleinsäuren im jeweiligen Expressionssystem zur Expression zu bringen. Geeignete Systeme werden beispielsweise in Current Protocols in Molecular Biology, F. Ausubel et al., Hrsg., Wiley Interscience, New York 1997, oder Sambrook et al. Molecular Cloning: A Laboratory Manual. 2. Aufl., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 beschrieben.The Introduce of DNA molecules in cells, familiar cloning methods known to those skilled in the art are used. and transfection methods, such as co-precipitation, Protoplast fusion, electroporation, retroviral transfection and the like, used to bind said nucleic acids in respective expression system for expression. suitable Systems are used, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Eds., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
Weiterhin kann das Einführen von DNA in Zellen auch durch homologe Rekombination erfolgen. Dazu wird die DNA üblicherweise in einen entsprechenden Vektor zur homologen Rekombination eingebracht, wie er beispielsweise aus Thomas, K.R. und Capecchi, M.R. (1987) Cell 51:503 bekannt ist.Farther can the introduction from DNA into cells also by homologous recombination. This will be the DNA usually introduced into an appropriate vector for homologous recombination, as he is known, for example, Thomas, K.R. and Capecchi, M.R. (1987) Cell 51: 503 is known.
Das Züchten der Zellen geschieht je nach Wirtsorganismus in dem Fachmann bekannter Weise. Mikroorganismen werden in der Regel in einem flüssigen Medium, das eine Kohlenstoffquelle meist in Form von Zuckern, eine Stickstoffquelle meist in Form von organischen Stickstoffquellen wie Hefeextrakt oder Salzen wie Ammoniumsulfat, Spurenelemente wie Eisen-, Mangan-, Magnesiumsalze und gegebenenfalls Vitamine enthält, bei Temperaturen zwischen 0 °C und 100 °C, bevorzugt zwischen 10 °C bis 60 °C unter Sauerstoffbegasung angezogen. Dabei kann der pH der Nährflüssigkeit auf einen festen Wert gehalten werden, das heißt während der Anzucht reguliert werden oder nicht. Die Anzucht kann „batch"-weise, „semi batch"-weise oder kontinuierlich erfolgen. Nährstoffe können zu beginn der Fermentation vorgelegt oder semikontinuierlich oder kontinuierlich nachgefüttert werden.The Breed The cells are known in the art depending on the host organism Wise. Microorganisms are usually in a liquid medium, the one carbon source mostly in the form of sugars, a nitrogen source mostly in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, Magnesium salts and optionally contains vitamins, at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C up to 60 ° C attracted under oxygen fumigation. In this case, the pH of the nutrient fluid be kept at a fixed value, that is regulated during the cultivation be or not. The cultivation can be batchwise, semi-batchwise or continuous respectively. nutrient can presented at the beginning of the fermentation or semicontinuously or continuously fed in become.
Auswählen von Zellen, die eine Laccase aus Aspergillus nidulans exprimieren geschieht durch mittels des Laccase katalysierten Umsatz eines Substrats, das danach als leicht identifizierbares Produkt, üblicherweise als Farbstoff vorliegt. Solche gefärbten Zellen bzw. Kolonien, die in ihrer Umgebung das Substrat im Agar umsetzen, können leicht von den nicht-gefärbten Zellen bzw. Kolonien unterschieden und anschliessend separiert werden.Select from Cells that express a laccase from Aspergillus nidulans happens by laccase catalyzed conversion of a substrate, then as easily identifiable product, usually is present as a dye. Such colored cells or colonies, those in their environment to implement the substrate in agar, can easily from the non-colored Distinguish cells or colonies and then separated.
Besonders geeignete Substrate sind die Verbindungen ABTS (2,2'-azino-bis-(3-ethylbenzthiazolin-6-sulfonate), DMA (3,5-dimethylaniline) und ADBP (4-amino-2,6-dibromophenol). Diese Verbindungen werden im folgenden auch mit dem Begriff „Testsubstrate" bezeichnet.Especially suitable substrates are the compounds ABTS (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonate), DMA (3,5-dimethylaniline) and ADBP (4-amino-2,6-dibromophenol). These connections will be hereinafter also referred to by the term "test substrates".
Diese Testsubstrate können von Laccasen in Gegenwart von Sauerstoff zu einem blauen Farbstoff umgesetzt werden, wodurch eine schnelle Erkennung von Laccase-Aktivität möglich wird. Der Farbumschlag kann direkt mit dem Auge erkannt werden oder – insbesondere zur Quantifizierung – photometrisch gemessen werden.These Test substrates can converted from laccases in the presence of oxygen to a blue dye which enables rapid recognition of laccase activity. The color change can be recognized directly with the eye or - in particular for quantification - photometrically be measured.
Ein Vorteil bei einigen Ausführungsformen der erfindungsgemässen Verfahren ist die Tatsache, dass einige Laccasen aus A. nidulans aus der Zelle ins Medium sekretiert werden. Damit ist es möglich, die Laccase ausserhalb der Zelle, beispielsweise direkt auf der Agarplatte nachzuweisen, ohne dass die Testsubstrate in die Zelle aufgenommen werden müssen.One Advantage in some embodiments the inventive Method is the fact that some laccases from A. nidulans be secreted from the cell into the medium. This makes it possible for the Laccase outside the cell, for example directly on the agar plate to detect without the test substrates added to the cell Need to become.
Eine weitere bevorzugte Ausführungsform der erfindungsgemässen Verfahren besteht darin, dass die einzelnen Laccasen verschiedene Spezifitäten gegenüber den Testsubstraten aufweisen. Somit wird es möglich, differenzielle Untersuchungen mit beispielsweise zwei Laccasen durchzuführen, wobei die erste Laccase das Testsubstrat 1 und nicht das Testsubstrat 2 umsetzt und die zweite Laccase das Testsubstrat 2 und nicht das Testsubstrat 1 umsetzt. Die in der untenstehenden Tabelle angegebene Substratspezifität der Laccasen lccB, lccC, und lccD erlaubt die Auswahl geeigneter Laccasen und Testsubstrate für ein solches differentielles Verfahren.A further preferred embodiment the inventive Method is that the individual laccases different specificities across from have the test substrates. Thus, it becomes possible to differential investigations to carry out with, for example, two laccases, wherein the first laccase the Test substrate 1 and not the test substrate 2 converts and the second Laccase the test substrate 2 and not the test substrate 1 converts. The substrate specificity of the laccases lccB given in the table below, lccC, and lccD allows the selection of suitable laccases and test substrates for a such a differential method.
Für die erfindungsgemässen Verfahren geeignete Laccasen sind die in Aspergillus nidulans natürlicherweise vorkommenden Laccasen einschliesslich von Polymorphismen, Allelen und Sequenzvarianten, sowie die davon ableitbaren Laccasen, soweit sie noch die wesentlichen biologischen Eigenschaften der natürlich vorkommenden Lac casen, insbesondere die Substratspezifität gegenüber den oben genannten Testsubstraten, aufweisen.For the inventive method suitable laccases are those in Aspergillus nidulans naturally occurring laccases including polymorphisms, alleles and sequence variants, as well as the derivable laccases, as far as They still have the essential biological properties of naturally occurring Lac casen, in particular the substrate specificity with respect to the test substrates mentioned above, exhibit.
Besonders
geeignete Laccasen sind die Laccasen lccA, lccB, lccC und lccD aus
A. nidulans, deren DNA- und Polypeptidsequenzen im Sequenzprotokoll
aufgeführt
sind.
Eine besonders vorteilhafte Ausgestaltung der erfindungsgemässen Verfahren ergibt sich dadurch, dass die Laccasen aus A. nidulans verschiedene Spezifitäten gegenüber den Testsubstraten besitzen. Die folgende Tabelle gibt die Substratspezifitäten wieder: A particularly advantageous embodiment of the process according to the invention results from the fact that the A. nidulans laccases have different specificities compared to the test substrates. The following table shows the substrate specificities:
- + : setzt das genannte Substrat mit Sauerstoff zu einem blauen Farbstoff um+: sets the said substrate with oxygen to a blue dye around
- – : keine Umsetzung mit Sauerstoff zu einer farbigen Verbindung-: no conversion with oxygen to a colored compound
Weiter geeignete Farbstoffe zum Laccasenachweis sind die von Dias et al., 2003 und Johannes & Majcherezyk, 2000 beschriebenen.Further suitable dyes for laccase detection are those of Dias et al., 2003 and Johannes & Majcherezyk, 2000 described.
Experimenteller Teilexperimental part
Isolierung von Laccasen aus Aspergillus nidulansinsulation of laccases from Aspergillus nidulans
Das Genom von A. nidulans wurde auf das Vorkommen von Laccasen untersucht und neben YA und TilA vier neue Laccasen, lccA, lccB, lccC und lcc-D, entdeckt. Die Sequenz der gefundenen Laccasen ist im Sequenzprotokoll dargestellt.The Genome of A. nidulans was examined for the presence of laccases and next to YA and TilA four new laccases, lccA, lccB, lccC and lcc-D, discovered. The sequence of the laccases found is in the sequence listing shown.
Die Laccasen lccB, lccC und lccD wurden mittels PCR amplifiziert und unter der Kontrolle des durch Alkohole induzierbaren Promotors, alcA, sowie den xylA Promotor, in A. nidulans exprimiert. (Waring et al. 1989, Zadra et al. 2000).The Laccases lccB, lccC and lccD were amplified by PCR and under the control of the alcohol-inducible promoter, alcA, as well as the xylA promoter, expressed in A. nidulans. (Waring et al. 1989, Zadra et al. 2000).
Zum Test der Enzymaktivität wurden die Stämme auf threoninhaltigen oder xylosehaltigen Agarplatten kultiviert und die Laccase katalysierte Oxidation von ABTS (2,2'-azino-bis-(3-ethylbenzthiazolin-6-sulfonate) untersucht.To the Test of enzyme activity were the tribes cultivated on threonine-containing or xylose agar plates and the laccase catalyzed oxidation of ABTS (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonates) studied.
Im
Falle von LccC kam es zu einem Farbumschlag (
Laccasen als Reportergene zur Bestimmung von PromotoraktivitätenLaccases as Reporter genes for the determination of promoter activities
Der offene Leserahmen von Laccase C kann mit einem Promotor fusioniert werden – analog zu der Fusion mit dem alcA oder xylA Promotor – und dadurch dessen Aktivität bestimmt werden. Ein Pilz kann unter verschiedenen Bedingungen (Temperatur, pH, Mediumzusammensetzung) kultiviert werden und damit die Bedingungen der Promotoraktivität ermittelt werden. Der Pilz kann auf Agarplatten kutliviert werden und die Aktivität visuell oder spektroskopisch beurteilt werden. Ausserdem kann der Pilz in Flüssigkultur gezüchtet und die Laccaseaktivität im Kulturüberstand quantifiziert werden. Laccase B und D können entsprechend mit den beiden anderen Substraten eingesetzt werden.Of the open reading frame of laccase C can be fused with a promoter be - analog to the fusion with the alcA or xylA promoter - thereby determining its activity become. A fungus can be stored under different conditions (temperature, pH, medium composition) and thus the conditions the promoter activity be determined. The fungus can be cut on agar plates and the activity assessed visually or spectroscopically. In addition, the Mushroom in liquid culture cultured and the laccase activity in culture supernatant be quantified. Laccase B and D can be used with both other substrates are used.
Laccasen zur Lokalisierung von ProteinenLaccases to Localization of proteins
Durch eine translationale Fusion eines N-terminalen Bereichs eines zu testenden Proteins mit einer Laccase, in der das Sekretionssignal deletiert wurde, kann die Funktionalität eines vorhergesagten Sekretionssignals getestet werden.By a translational fusion of an N-terminal region of an testing protein with a laccase, in which the secretion signal has deleted the functionality of a predicted secretion signal be tested.
Durch eine translationale Fusion eines zu testenden Proteins mit der Laccase (mit deletiertem Sekretionssignal) kann die subzelluläre Lokalisierung des Proteins untersucht werden. Zellorganellen können fraktioniert werden und die Laccaseaktivität in den verschiedenen Fraktionen getestet werden.By a translational fusion of a protein to be tested with the laccase (with deleted secretion signal) may be the subcellular localization of the protein. Cell organelles can be fractionated and the laccase activity be tested in the different fractions.
Laccasen als Reportergen für genetische ScreensLaccases as Reporter gene for genetic screens
Zur Isolierung von Promotoren, die auf bestimmte Umweltparameter (Temperatur, pH, Substrate) reagieren, kann LccC mit DNA-Fragmenten fusioniert werden. Die erstellte Genbank kann in einen Pilzstamm transformiert werden und positive, d.h. Laccasesekretierende Stämme können anhand der Blaufärbung isoliert werden. Dieser Ansatz kann auch in Mikrotiterplatten durchgeführt werden, so dass der "Readout" durch Roboter erfolgen kann. Damit ist die Methode auch für Hoch-Durchsatz-Experimente geeignet.to Isolation of promoters based on specific environmental parameters (temperature, pH, substrates), LccC can be fused with DNA fragments become. The generated gene bank can be transformed into a fungus strain and positive, i. Laccase-secreting strains can be identified the blue coloration be isolated. This approach can also be performed in microtiter plates, so that the "readout" by robots done can. Thus, the method is also for high-throughput experiments suitable.
Laccasen können auch zur Isolierung von Regulatoren definierter Promotoren dienen. Dazu wird ein bestimmter Promotor mit dem Laccasegen fusioniert und in einen Pilzstamm eingebracht. Der Stamm, der dann Expression der Laccase entsprechend der Promotoraktivität zeigt, kann mutagenisiert werden, um Mutanten mit einer veränderten Regulation zu isolieren. Dadurch können z.B. Stämme mit verstärkter Promotoraktivität hergestellt werden. Wenn der gleiche Promotor in dem Stamm zur Expression eines gewünschten Gens genutzt wird, kann so die Expression dieses Gens gesteigert werden. Es können auch Mutanten mit verringerter Prommotorstärke isoliert werden. Dieser Effekt kann darauf beruhen, dass positive Regulatoren mutiert wurden. Diese Mutanten können genutzt werden, um die entsprechenden Regulatoren z.B. durch Komplementation mit einer Genbank zu isolieren.laccases can also serve for the isolation of regulators defined promoters. For this purpose, a specific promoter is fused with the laccase gene and introduced into a mushroom strain. The trunk, then expression the laccase according to the promoter activity can be mutagenized to isolate mutants with altered regulation. Thereby can e.g. strains with reinforced promoter activity getting produced. When the same promoter in the strain for expression a desired gene is used, so the expression of this gene can be increased. It can also mutants with reduced prommotor strength can be isolated. This Effect may be due to the fact that positive regulators have been mutated. These mutants can be used to control the corresponding regulators e.g. through complementation to isolate with a gene bank.
Laccasen lccB, lccC oder lccD als Reportergene zur Quantifizierung von PromotoraktivitätenLaccases lccB, lccC or lccD as reporter genes for the quantification of promoter activities
Der offene Leserahmen der drei Laccasen, in dem die Sekretionssignale deletiert wurden, können mit Promotoren fusioniert und in Pilze transformiert werden. Diese Konstrukte führen zur Produktion von intrazellulärer Laccase. Die Enzymaktivität kann nach Zellaufschluss exakt quantifiziert werden. Da die drei Laccasen unterschiedliche Substrate umsetzen, können mehrere Promotoraktivitäten gleichzeitig quantifiziert werden. Dieses System ähnelt dem simultanen Einsatz von beta-Galaktosidase und Glucuronidase.Of the open reading frame of the three laccases, in which the secretion signals could have been deleted be fused with promoters and transformed into fungi. These Constructs lead for the production of intracellular Laccase. The enzyme activity can be quantified exactly after cell disruption. Because the three Laccases can implement different substrates, allowing multiple promoter activities simultaneously be quantified. This system is similar to simultaneous use of beta-galactosidase and glucuronidase.
SEQUENCE LISTING SEQUENCE LISTING
Verzeichnis der Abbildungendirectory of the pictures
Literaturliterature
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