The green fluorescent protein gene functions as a reporter of gene expression in Phanerochaete chrysosporium.

The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter of gene expression driven by the glyceraldehyde-p-dehydrogenase (gpd) gene promoter and the manganese peroxidase isozyme 1 (mnp1) gene promoter in Phanerochaete chrysosporium. Four different constructs were prepared. pUGGM3' and pUGiGM3' contain the P. chrysosporium gpd promoter fused upstream of the egfp coding region, and pUMGM3' and pUMiGM3' contain the P. chrysosporium mnp1 promoter fused upstream of the egfp gene. In all constructs, the egfp gene was followed by the mnp1 gene 3' untranslated region. In pUGGM3' and pUMGM3', the promoters were fused directly with egfp, whereas in pUGiGM3' and pUMiGM3', following the promoters, the first exon (6 bp), the first intron (55 bp), and part of the second exon (9 bp) of the gpd gene were inserted at the 5' end of the egfp gene. All constructs were ligated into a plasmid containing the ura1 gene of Schizophyllum commune as a selectable marker and were used to transform a Ural1 auxotrophic strain of P. chrysosporium to prototrophy. Crude cell extracts were examined for GFP fluorescence, and where appropriate, the extracellular fluid was examined for MnP activity. The transformants containing a construct with an intron 5' of the egfp gene (pUGiGM3' and pUMiGM3') exhibited maximal fluorescence under the appropriate conditions. The transformants containing constructs with no introns exhibited minimal or no fluorescence. Northern (RNA) blots indicated that the insertion of a 5' intron resulted in more egfp RNA than was found in transformants carrying an intronless egfp. These results suggest that the presence of a 5' intron affects the expression of the egfp gene in P. chrysosporium. The expression of GFP in the transformants carrying pUMiGM3' paralled the expression of endogenous mnp with respect to nitrogen and Mn levels, suggesting that this construct will be useful in studying cis-acting elements in the mnp1 gene promoter.

Characterization of genes encoding two manganese peroxidases from the lignin-degrading fungus Dichomitus squalens(1).

Genes encoding two manganese peroxidases from the white-rot basidiomycete Dichomitus squalens were cloned and sequenced. The mnp1 and mnp2 genes encode mature proteins of 369 and 365 amino acids, respectively. The amino acids involved in peroxidase function, those forming the Mn(II) binding site, and those forming the five disulfide bonds in other Mn peroxidases are conserved in these sequences. Both predicted D. squalens proteins contain multiple acidic residues in their C-terminal sequences, which may be involved in additional metal binding. Both genes contain seven small introns, the locations of which align with each other. The promoters of both D. squalens genes contain putative AP-2 sites, which may be involved in their regulation by nutrient nitrogen. Southern blot analysis of genomic PCR fragments suggests that these sequences represent separate genes rather than allelic variants.

Characterization of manganese(II) binding site mutants of manganese peroxidase.

A series of site-directed mutants, E35Q, E39Q, and E35Q-D179N, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium, was created by overlap extension, using the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidases (MnPs) were purified and characterized. The molecular masses of the mutant proteins, as well as UV-vis spectral features of their oxidized states, were very similar to those of the wild-type enzyme. Resonance Raman spectral results indicated that the heme environment of the mutant MnP proteins also was similar to that of the wild-type protein. Steady-state kinetic analyses of the E35Q and E39Q mutant MnPs yielded K(m) values for the substrate MnII that were approximately 50-fold greater than the corresponding K(m) value for the wild-type enzyme. Likewise, the kcat values for MnII oxidation were approximately 300-fold lower than that for wild-type MnP. With the E35Q-D179N double mutant, the K(m) value for MnII was approximately 120-fold greater, and the kcat value was approximately 1000-fold less than that for the wild-type MnP1. Transient-state kinetic analysis of the reduction of MnP compound II by MnII allowed the determination of the equilibrium dissociation constants (KD) and first- order rate constants for the mutant proteins. The KD values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were approximately 200-fold and approximately 4000-fold less, respectively, than that of the wild-type enzyme. In contrast, the K(m) values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. The second-order rate constants for p-cresol and ferrocyanide reduction of the mutant compounds II also were similar to those of the wild-type enzyme.

Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium.

The promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was used to drive expression of mnp1, the gene encoding Mn peroxidase isozyme 1, in primary metabolic cultures of Phanerochaete chrysosporium. A 1,100-bp fragment of the P. chrysosporium gpd promoter region was fused upstream of the mnp1 gene to construct plasmid pAGM1, which contained the Schizophyllum commune ade5 gene as a selectable marker. pAGM1 was used to transform a P. chrysosporium ade1 auxotroph to prototrophy. Ade+ transformants were screened for peroxidase activity on a solid medium containing high carbon and high nitrogen (2% glucose and 24 mM NH4 tartrate) and o-anisidine as the peroxidase substrate. Several transformants that expressed high peroxidase activities were purified and analyzed further in liquid cultures. Recombinant Mn peroxidase (rMnP) was expressed and secreted by transformant cultures on day 2 under primary metabolic growth conditions (high carbon and high nitrogen), whereas endogenous wild-type mnp genes were not expressed under these conditions. Expression of rMnP was not influenced by the level of Mn in the culture medium, as previously observed for the wild-type Mn peroxidase (wtMnP). The amount of active rMnP expressed and secreted in this system was comparable to the amount of enzyme expressed by the wild-type strain under ligninolytic conditions. rMnP was purified to homogeneity by using DEAE-Sepharose chromatography, Blue Agarose chromatography, and Mono Q column chromatography. The M(r) and absorption spectrum of rMnP were essentially identical to the M(r) and absorption spectrum of wtMnP, indicating that heme insertion, folding, and secretion were normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the mnp2 gene encoding manganese peroxidase isozyme 2 from the basidiomycete Phanerochaete chrysosporium.

The nucleotide (nt) sequence of a gene (mnp2) encoding manganese peroxidase isozyme 2 (MnP-2) from Phanerochaete chrysosporium was determined. The sequence of 3297 bp includes 1287 bp of 5'-flanking sequence and 490 bp 3' to the stop codon. Comparison of cDNA and genomic sequences indicates seven introns varying in size from 50-55 bp. The 5' upstream region of the mnp2 gene contains a TATAA element, three inverted CCAAT elements (ATTGG), six putative heat-shock elements (HSE) and three putative metal response elements (MRE) (TGCRCNC), all located within 1100-bp upstream from the start codon. The positions of the putative HSE and MRE in the promoter region of mnp2 are compared with the corresponding sequences in the mnp1 gene promoter. A Northern blot probed with a fragment specific for the mnp2 gene suggests that the transcription of mnp2 is regulated by Mn ions.

Characterization of a gene encoding a manganese peroxidase from Phanerochaete chrysosporium.

The complete nucleotide (nt) sequence of a gene (mnp-1) encoding manganese peroxidase isozyme 1 (MnP-1) (pI = 4.9) from Phanerochaete chrysosporium has been determined. The sequence of 2539 bp includes 526 bp of 5'-flanking sequence and 368 bp 3' to the poly(A) site. Comparison of cDNA and genomic sequences indicates six introns varying in size from 57-72 bp. Intron splice-junction sequences all adhere to the GT---AG rule. The positions of the introns show little similarity to the intron positions in the closely related lignin peroxidase-encoding genes. The 5' upstream region of the mnp-1 gene contains a TATAA element and three inverted CCAAT elements (ATTGG) at nt positions -81, -181, -195, and -304, respectively, relative to the start codon. In addition, the mnp-1 gene contains three putative heat-shock (HS) elements similar to the consensus C--GAA--TTC--G sequence, and two consensus metal response elements located within 500 bp upstream from the start codon. Furthermore, Northern-blot analysis demonstrates that mnp gene transcription is regulated by HS.

Characterization of a cDNA encoding a manganese peroxidase, from the lignin-degrading basidiomycete Phanerochaete chrysosporium.

A cDNA clone of a manganese peroxidase (MnP) from Phanerochaete chrysosporium was isolated and characterized. The cDNA contains 1314 nucleotides excluding the poly(A) tail and the coding region has 68% G + C content. The deduced mature MnP protein contains 357 amino acids and is preceded by a 21-amino acid leader sequence. The experimentally determined N-terminal sequence of the purified MnP-1 protein, pI = 4.9, corresponds to the deduced N-terminal sequence of the gene. The Mr of the mature MnP-1 deduced from the cDNA is 37,439, which is approximately 81.4% of the experimentally determined molecular weight. The difference is due to glycosylation and a single potential N-glycosylation site with the general sequence Asn-X-Thr/Ser is present in the deduced MnP-1 sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine, and the distal arginine are all conserved and regions flanking these residues display homology with other peroxidases. Northern blot analysis indicates that MnP expression is controlled by nutrient nitrogen at the level of transcription. Southern blot hybridization analysis suggests that MnP-1 is a member of a family of MnP genes.

Degradation of the gamma-Carboxyl-Containing Diarylpropane Lignin Model Compound 3-(4'-Ethoxy-3'-Methoxyphenyl)-2-(4''-Methoxyphenyl)Propionic Acid by the Basidiomycete Phanerochaete chrysosporium.

The white-rot basidiomycete Phanerochaete chrysosporium metabolized 3-(4'-ethoxy-3'-methoxyphenyl)-2-(4''-methoxyphenyl)propionic acid (V) in low-nitrogen, stationary cultures, conditions under which ligninolytic activity is expressed. The ability of several fungal mutant strains to degrade V reflected their ability to degrade [C]lignin to CO(2). 1-(4'-Ethoxy-3'-methoxyphenyl)-2-(4''-methoxyphenyl)-2- hydroxyethane (VII), anisyl alcohol, and 4-ethoxy-3-methoxybenzyl alcohol were isolated as metabolic products, indicating an initial oxidative decarboxylation of V, followed by alpha, beta cleavage of the intermediate (VII). Exogenously added VII was rapidly converted to anisyl alcohol and 4-ethoxy-3-methoxybenzyl alcohol. When the degradation of V was carried out under O(2), O was incorporated into the beta position of the diarylethane product (VII), indicating that the reaction is oxygenative.

An extracellular H2O2-requiring enzyme preparation involved in lignin biodegradation by the white rot basidiomycete Phanerochaete chrysosporium.

An H2O2-requiring enzyme system was found in the extracellular medium of ligninolytic cultures of Phanerochaete chrysosporium. The enzyme system generated ethylene from 2-keto-4-thiomethyl butyric acid (KTBA), and oxidized a variety of lignin model compounds including the diarylpropane 1-(4'-ethoxy-3'-methoxyphenyl) 1,3-dihydroxy-2-(4"-methoxyphenyl)propane (I), a beta-ether dimer 1-(4'-ethoxy-3'-methoxyphenyl)glycerol-beta-guaiacyl ether (IV) and an olefin 1-(4'-ethoxy-3'-methoxyphenyl)1,2-propene (VI). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures. In addition, the enzyme system partially degraded 14C-ring labeled lignin. The enzyme was not found in high nitrogen (N) cultures, nor in cultures of a ligninolytic mutant strain which is incapable of metabolizing lignin.

Isolation and Complementation Studies of Auxotrophic Mutants of the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium.

A variety of auxotrophic strains of Phanerochaete chrysosporium were isolated after treatment of conidia with UV and X rays. Complementation studies with these strains demonstrated heterokaryotic mycelia and conidia in this organism. Nuclear staining also showed that conidia can be mono-, di-, or multinucleate. Complementation tests allowed the separation of each auxotrophic class with the same phenotype into complementation groups.

Metabolism of Radiolabeled beta-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium.

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [gamma-C]guaiacylglycerol-beta-guaiacyl ether and [4-methoxy-C]veratrylglycerol-beta-guaiacyl ether (VI) to CO(2) in stationary and in shaking cultures. CO(2) evolution was greater in stationary culture. CO(2) evolution from [gamma-C]guaiacyl-glycerol-beta-guaiacyl ether and [4-methoxy-C]veratrylglycerol-beta-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O(2) rather than air (21% O(2)) was the gas phase above the cultures. CO(2) evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed CO(2) evolution from both substrates in stationary cultures. [C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-beta-guaiacyl ether, respectively.