Homologous transformation of the lignin-degrading basidiomycete Phanerochaete chrysosporium

Alic, Margaret; Mayfield, Mary B.; Akileswaran, Lakshmi; Gold, Michael H.

doi:10.1007/bf00312741

Cited by 55 publications

(50 citation statements)

References 34 publications

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“…No evidence for integration of the vector sequences into the chromosome was found even after 2 The results indicate that the ME-1 component of pG12-1 originated from a low-copy-number extrachromosomal plasmid (pME) of P. chrysosporium. Since the ME-1 component is common to both pG12-1 and pME, it appears possible that the ME-1 sequences acquired by pG12-1 might be responsible for the stability of this vector and its low-copy-number maintenance, which is similar to that of pME.…”

Section: Discussionmentioning

confidence: 80%

“…We previously described vector pRR12, which was shown to transform P. chrysosporium to G418 resistance and was rescued in Escherichia coli from the total DNA of pRR12 transformants (35). Alic et al (1,2) have described an integrative transformation system in which a plasmid vector carrying either an heterologous ADE2 or ADE5 gene from Schizophyllum commune was used to complement the appropriate adenine auxotroph of P. chrysosporium. We describe here a novel transformation vector for P. chrysosporium that is maintained stably in the transformants in an extrachromosomal circular form and is readily recoverable from the total DNA of transformants.…”

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confidence: 99%

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A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium

1991

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A stable extrachromosomally maintained transformation vector (pG12-1) for the lignin-degrading ifiamentous fungus Phanerochaete chrysosporium is described. The vector is 6.3 kb and contains a Kanr marker, pBR322 on, and a 2.2-kb fragment (ME-1) derived from an endogenous extrachromosomal DNA element of P. chrysosporium. Vector pG12-1 was able to transform P. chrysosporium to G418 resistance and was readily and consistently recoverable from the total DNA of transformants via Escherichia coli transformation. Southern blot analyses indicated that pG12-1 is maintained at a low copy number in the fungal transformants. The vector is demonstrable in the total DNA of individual G418-resistant basidiospore progeny of the transformants only after amplification by polymerase chain reaction. Exonuclease IH and dam methylation analyses, respectively, indicated that pG12-1 undergoes replication in P. chrysosporium and that it is maintained extrachromosomally in a circular form. The vector is stably maintained in the transformants even after long-term nonselective growth. There is no evidence for integration of the vector into the chromosome at any stage.In recent years, research has intensified worldwide on the ligninolytic fungus Phanerochaete chrysosporium because of the industrial potential of this organism in biopulping, in the conversion of lignin and lignocellulosic materials to feeds, fuels, and chemicals (22, 47), and in detoxifying recalcitrant environmental pollutants such as dioxins, polychlorinated biphenyls, and benzo(a)pyrenes (10, 11). Lignin peroxidases and manganese peroxidases, two families of extracellular, glycosylated, heme proteins involved in lignin degradation by P. chrysosporium, have been isolated and characterized (16, 22,47,48). Genetic studies with this organism have been limited, but auxotrophic mutants (15,26,27) and mutants lacking various secondary metabolic activities (7, 21, 24) have been isolated. Both cDNA (13,49,53,55) and genomic clones (3,5,8,20,41,42,52,54) for lignin peroxidases and cDNA clones for manganese peroxidases (31, 34) have been isolated and sequenced. We previously described vector pRR12, which was shown to transform P. chrysosporium to G418 resistance and was rescued in Escherichia coli from the total DNA of pRR12 transformants (35). Alic et al. (1, 2) have described an integrative transformation system in which a plasmid vector carrying either an heterologous ADE2 or ADE5 gene from Schizophyllum commune was used to complement the appropriate adenine auxotroph of P. chrysosporium. We describe here a novel transformation vector for P. chrysosporium that is maintained stably in the transformants in an extrachromosomal circular form and is readily recoverable from the total DNA of transformants. MATERIALS AND METHODSStrains and media. P. chrysosporium ME446 (ATCC 34541) was maintained as previously described (21). E. coli DHSa [F-endAl hsdR17(rK-MK+) supE44 thi-J deoR recAl gyrA96 relAIA (argF-lacZYA)U169 4i80dlacZAMJSA-], was used for the maintenance of plasmid vectors. Gold...

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Section: Discussionmentioning

confidence: 80%

mentioning

confidence: 99%

A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium

1991

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“…Conidia from prototrophs were then cultured in high carbon high nitrogen (HCHN) stationary liquid cultures with glucose as the sole carbon source (8,19) and assayed for extracellular CDH activity using both the cyt c and DCPIP assays (8,19). The transformant exhibiting the highest activity was purified by isolating single basidiospores as described elsewhere (21,22), and progeny were rescreened for CDH activity in liquid cultures.…”

Section: Methodsmentioning

confidence: 99%

Biophysical and Structural Analysis of a Novel Heme b Iron Ligation in the Flavocytochrome Cellobiose Dehydrogenase

Rotsaert

Hällberg

Vries

et al. 2003

Journal of Biological Chemistry

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Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 Å resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N ␦1 and N ⑀2 nitrogen atoms, rather than the typical N ⑀2 /N ⑀2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N ␦1 /N ⑀2 -coordinated protoporphyrin IX iron.

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“…Culture conditions. Stock cultures of P. chrysosporium (OGC101) (Alic et al, 1987) and D. squalens (Karst Reid) (CBS 432.34) (Perie & Gold, 1991) were maintained on slants as described previously (Alic et al, 1987;PCrie & Gold, 1991). Liquid cultures were grown from a conidial inoculum at 38 OC (P. chrysosporium) or from a mycelial inoculum at 28 "C (D.…”

Section: Methodsmentioning

confidence: 99%

“…squalens) in stationary culture (25 ml; 250 ml flask) as described previously (Alic et al, 1987;E r i e & Gold, 1991). The medium, described previously (Kirk et al, 1978;Perie & Gold, 1991) (Rieble et al, 1994;Brock et al, 1995).…”

Section: Methodsmentioning

confidence: 99%

Degradation of chlorophenoxyacetic acids by the lignin-degrading fungus Dichomitus squalens

Reddy¹,

Joshi²,

Gold³

1997

Microbiology

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We have examined the degradation of 14C ring-and side-chain-labelled 2,4,5-trichlorophenoxyacetic acid by Dichomitus squalens and Phanerochaete chrysosporium. The effects of Mn2+ on the degradation of these radiolabelled substrates by D. squalens and the effect of nitrogen limitation on their degradation by P. chrysosporium suggested that in both fungi, side-chain cleavage was catalysed by a mechanism independent of the lignin degradation system, whereas the degradation of the aromatic ring was dependent on the lignin degradative system. Using unlabelled substrates, a pathway for the degradation of chlorophenoxyacetic acids was elucidated in D. squalens. Time courses for the degradation of unlabelled chlorophenoxyacetic acids by D. squalens demonstrated that the corresponding chlorophenol was the initial product formed. The chlorophenol intermediate was xylosylated to form the chlorophenolxyloside. In turn, the chlorophenolxyloside could be hydrolysed by an intracellular p-xylosidase to regenerate the chlorophenol. The chlorophenol product of the xylosidase reaction was oxidatively dechlorinated to form 2-chloro-p-benzoquinone which could undergo subsequent further dechlorination and ring-opening reactions, as has been reported previously for P. chrysosporium.

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Homologous transformation of the lignin-degrading basidiomycete Phanerochaete chrysosporium

Cited by 55 publications

References 34 publications

A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium

A novel extrachromosomally maintained transformation vector for the lignin-degrading basidiomycete Phanerochaete chrysosporium

Biophysical and Structural Analysis of a Novel Heme b Iron Ligation in the Flavocytochrome Cellobiose Dehydrogenase

Degradation of chlorophenoxyacetic acids by the lignin-degrading fungus Dichomitus squalens

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