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The promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was used to drive expression of mnpl, 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 mnpl gene to construct plasmid pAGMI, which contained the SchizophyUlum commune adeS gene as a selectable marker. pAGM1 was used to transform a P. chrysosporium adel 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 Mr and absorption spectrum of rMnP were essentially identical to the Mr and absorption spectrum of wtMnP, indicating that heme insertion, folding, and secretion were normal. The steady-state kinetic values for the oxidation of Mn(II) and 2,6-dimethoxyphenol by rMnP and wtMnP also were very similar. This system is suitable for generating

The expression of manganese peroxidase in nitrogen-limited cultures of Phanerochaete chrysosporium is dependent on Mn, and initial work suggested that Mn regulates transcription of the mnp gene. In this study, using Northern (RNA) blot analysis of kinetic, dose-response, and inhibitor experiments, we demonstrate unequivocally that Mn regulates mnp gene transcription. The Lignin, the most abundant aromatic polymer, is a complex, optically inactive phenylpropanoid matrix that constitutes 15 to 30% of woody plant cell walls (10, 41). White rot basidiomycetes are primarily responsible for the initiation of the decomposition of lignin in wood (8, 20, 26). The beststudied lignin-degrading basidiomycete, Phanerochaete chrysosporium, degrades lignin during the secondary metabolic (idiophasic) phase of growth, which is triggered by limiting cultures for nutrient nitrogen (20,26). Under ligninolytic conditions, P. chrysosporium secretes two extracellular heme peroxidases-manganese peroxidase (MnP) and lignin peroxidase (LiP)-which, along with an H202-generating system, are apparently the major components of its lignin degradation system (8,20,26). The structure and mechanism of LiP have been examined extensively (20,22,26,42), and cDNA (12, 48) and genomic clones (4, 46, 49) encoding several LiP isozymes have been characterized.MnP was discovered in our laboratory (28) and has been purified and characterized (16,17,20,28,37,50,51 MATERIALS AND METHODSCulture conditions. P. chrysosporium OGC101 (3) was maintained on slants as previously described (19). The organism was grown at 38°C from a conidial inoculum in 20-ml stationary cultures in 250-ml Erlenmeyer flasks as described previously (13). Cultures were incubated under air for 2 days, after which they were purged daily with 100% 02-The medium was as previously described (7,27), with 2% glucose as the carbon source, 1.2 mM ammonium tartrate as the limiting nitrogen source, 20 mM sodium-2,2-dimethylsuccinate (pH 4.5) as the buffer, and a modified trace elements solution (7)

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Prototrophic strains recovered from crosses between auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium were induced to fruit. The progeny of most of these self-crosses were prototrophic, indicating that the nuclei of the original prototroph were wild-type recombinants rather than complementary heterokaryons and that the binucleate basidiospores of this organism are homokaryotic. Various wild-type strains were shown to have multinucleate cells lacking clamp connections and to possess a variable number of sterigmata per basidium. Colonies arising from single conidia of various wild-type strains were all capable of producing fruit bodies and basidiospores. In addition, single basidiospores from three wild-type strains all produced fruit bodies and basidiospores. Nonfruiting as well as fruiting isolates were obtained from single basidiospores of five other wild-type strains. Basidiospores from these fruiting isolates always yielded colonies that fruited, again indicating that the spores are homokaryotic. Nonfruiting isolates from the same strain did not produce basidiospores when allowed to form a heterokaryon, implying that these isolates do not represent mating types. All this evidence indicates that P. chrysosporium has a primary homothallic mating system. In addition to fruiting and nonfruiting phenotypes, basidiospores from strain OGC101, a derivative of ME-446, gave rise to colonies which did not grow on cellulose (Cel-). The fruiting, nonfruiting, and Celphenotypes differed from each other and from the parental wild-type strain in a variety