Browsing by Author "Cullen, D"

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    Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78
    (2004) Martinez, D; Larrondo, LF; Putnam, N; Gelpke, MDS; Huang, K; Chapman, J; Helfenbein, KG; Ramaiya, P; Detter, JC; Larimer, F; Coutinho, PM; Henrissat, B; Berka, R; Cullen, D; Rokhsar, D
    White rot fungi efficiently degrade lignin, a complex aromatic polymer in wood that is among the most abundant natural materials on earth. These fungi use extracellular oxidative enzymes that are also able to transform related aromatic compounds found in explosive contaminants, pesticides and toxic waste. We have sequenced the 30-million base-pair genome of Phanerochaete chrysosporium strain RP78 using a whole genome shotgun approach. The P. chrysosporium genome reveals an impressive array of genes encoding secreted oxidases, peroxidases and hydrolytic enzymes that cooperate in wood decay. Analysis of the genome data will enhance our understanding of lignocellulose degradation, a pivotal process in the global carbon cycle, and provide a framework for further development of bioprocesses for biomass utilization, organopollutant degradation and fiber bleaching. This genome provides a high quality draft sequence of a basidiomycete, a major fungal phylum that includes important plant and animal pathogens.
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    Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns
    (2003) Larrondo, LF; Avila, M; Salas, L; Cullen, D; Vicuña, R
    Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described Ics-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active laccase. Relative to the isozymic forms of the native C. subvermispora enzyme, the A. niger-produced laccase had a higher molecular mass and gave a single band on IEF gels. In contrast, A. nidulans transformants secreted several isoforms remarkably similar to those of the native system. Considered together with previously reported Southern blots and protein sequencing, expression in A. nidulans supports the view that C. subvermispora has a single laccase gene and that multiple isoforms result from post-translational processes. In addition, several lines of evidence strongly suggest that under copper limitation, A. nidulans secretes apoprotein which can be reconstituted by a short incubation with Cu(I) and to a lesser extent with Cu(II).
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    Isoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium
    (2001) Larrondo, LF; Lobos, S; Stewart, P; Cullen, D; Vicuña, R
    We expressed cDNAs coding for manganese peroxidases (MnPs! from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha -amylase promoter from. Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar Levels and had molecular masses, both before and after deglycosylation, that were the same as those described For the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pls between 4.83 and 4.06, and one of these pls coincided with the pi described for H4 isolated from P. chrysosporium (pl 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.
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    Isolation and characterization of homokaryotic strains from the ligninolytic basidiomycete Ceriporiopsis subvermispora
    (2001) Tello, M; Seelenfreund, D; Lobos, S; Gaskell, J; Cullen, D; Vicuña, R
    Genetic analyses of the lignin-degrading fungus Ceriporiopsis subvermispora is complicated by a dikaryotic nuclear condition and the absence of sport: forms. Previous investigations had identified a family of closely related sequences encoding manganese peroxidase (MnP), but the relationship between genes and allelic variants could not be experimentally established. Addressing this issue, homokaryotic derivatives of C. subvermipora strain FP105752 were isolated from regenerated protoplasts. Designated CsA and CsB. their homokaryotic nature was established by polymerase chain reaction amplification and sequence analysis of the allelic variants of three MnP genes. Isoelectrofocusing revealed fewer MnP isoenzymes in filtrates of homokaryon cultures relative to the parental strain. The homokaryotic strains will simplify genetic analyses, particularly the identification of neu genes. (C) 2001 Federation of European Microbiological Societies. published by Elsevier Science B.V. All rights reserved.
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    lip-like genes in Phanerochaete sordida, and Ceriporiopsis subvermispora, white rot fungi with no detectable lignin peroxidase activity
    (1996) Rajakumar, S; Gaskell, J; Cullen, D; Lobos, S; Karahanian, E; Vicuna, R
    Lignin peroxidase-like genes were PCR amplified from Phanerochaete sordida and Ceriporiopsis subvermispora, fungi lacking lignin peroxidase (LiP) activity. Amplification products were highly similar to previously described LiP genes. Using reverse transcription-coupled PCR a LiP-like cDNA clone was amplified from P. sordida RNA. In contrast, no evidence was obtained for transcription of C. subvermispora LiP genes.