Heterologous gene expression and transcriptional regulation of manganese peroxidase gene expression in the basidiomycete Phanerochaete chrysosporium

Lignin peroxidase (LiP) and manganese peroxidase (MnP) are the major components of the lignin degradation system in the white-rot fungus Phanerochaete chrysosporium. Expression of P. chrysosporium LiP-encoding genes and MnP-encoding genes (mnp) is activated by nutrient nitrogen depletion. Moreover, expression of mnp requires the presence of Mn2+. Both nitrogen and Mn2+ levels regulate mnp expression at the transcriptional level.; The enhanced green fluorescent protein (GFP) gene (egfp) was used as a reporter in P. chrysosporium for gene expression driven by the glyceraldehyde-pdehydrogenase gene (gpd) promoter and the MnP isozyme 1 gene (mnp1) promoter. The 1.1-kb gpd promoter or the 1.5-kb mnp1 promoter was joined with the 5′ end of the egfp coding region, while the 250-bp mnp1 3′ untranslated region (UTR) was joined with the 3′ end of the egfp coding region. Efficient expression of egfp was only observed when an additional intron-containing sequence was inserted in the 5′ end of the egfp transcription unit. The GFP fluorescence levels faithfully reported the activity of both the gpd and mnp1 promoters. The 1.5-kb mnp1 promoter was sufficient for promoting egfp expression only under nitrogen-limited and Mn2+-sufficient conditions, similar to that required for endogenous MnP expression. A series of deletion, replacement and translocation mutations were introduced in this promoter, and their effects on egfp expression in response to Mn2+ status were examined. A 33-bp cis-element residing 521 by upstream of the translation start codon in the mnp1 promoter was identified that participates in the Mn2+-dependent regulation of mnp1 promoter activity. Negative control is exerted on this promoter element under Mn2+-deficient conditions. All mutations constructed in the 1.5-kb mnp1 promoter did not affect nitrogen-dependent gene regulation.; An efficient transformation system for P. chrysosporium was developed using the bacterial Bialaphos resistance gene (bar) as a selectable marker. The plasmid containing the bar coding region joined with the gpd promoter and the mnp1 3′ UTR onfers resistance to phosphinothricin (the active component of Bialaphos) when introduced into the P. chrysosporium wild-type strain OGC101. An efficient homologous expression system for P. chrysosporium MnP isozyme 1 was constructed using bar as a selectable marker.