A reporter gene construct for studying the regulation of manganese peroxidase gene expression

Abstract

The orotidylate decarboxylase (ODase) gene (ural) from Schizophyllum commune was utilized as a reporter for studying Mn regulation of the manganese peroxidase (MnP) gene (mnp) from the lignin-degrading basidiomycete Phanerochaete chrysosporium. A 1,500-bp fragment of the mnpl promoter was fused upstream of the coding region of the ODase gene in a plasmid (pAMO) containing the S. commune adeS gene as a selectable marker. pAMO was used to transform a P. chrysosporium adel urall mutant lacking endogenous ODase activity. When the P. chrysosporium transformant was grown in nitrogen-limited, Mn(II)-sufficient cultures, ODase activity was detected only during secondary metabolic growth and the pattern of ODase expression was similar to that of endogenous MnP. When Mn was added to 6-day-old nitrogen-limited, Mn-deficient cultures, both ODase activity and MnP activity were induced synchronously with maximal activity at 30 h. Growth in high-nitrogen-concentration medium suppressed the induction of both the ODase and endogenous MnP. These results indicate that this promoter-reporter construct can be used to study the regulation of the mnp gene.

Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium

“…The MnP activity reached a maximum level after 60 h and decreased thereafter. No new activity peak appeared on day 5 or 6 in any of the cultures; this is the period during which endogenous MnP is expressed in low-nitrogen cultures (5,11). These observations indicated that the activity observed in the transformants was due to rMnP.…”

“…MnP is a component of the lignin degradation systems of most, if not all, white rot fungi (19,32,35), including P. chrysosporium, in which it is expressed during idiophasic growth (10,11,14,38). MnP is a unique peroxidase in that its primary substrate is Mn(II), which is oxidized to Mn(III), and MnP activity is stimulated by various organic acid chelators, especially oxalate, which is secreted by P. chrysosporium.…”

“…Previously, we developed a DNA transformation system for P. chrysosporium which was based on complementation of auxotrophic mutants by heterologous or homologous biosynthetic genes (1,3). We recently utilized this system to study the expression of a reporter gene under the control of the mnp gene promoter (11). In this paper we describe homologous expression of mnpl under the control of the gpd promoter.…”

“…1A). The XbaI-NotI insert fragmeni tained the gpd promoter and linker, and a 3.6-ki fragment of the mnpl genomic clone (12), includi mnpl coding region from nucleotide 39, were use three-way ligation with the pOGI18 shuttle vect4 the Schizophyllum commune adeS gene as a selec (11) to give pAGM1 (Fig. 1B).…”

A reporter system for analysis of regulatable promoter functions in the basidiomycete fungus Phanerochaete chrysosporium

“…© 1998 The Society for Applied Microbiology have been used for gene replacement ) and for production of recombinant manganese peroxidase in P. chrysosporium (Mayfield et al 1994). In addition, they have been employed to develop a reporter gene construct for studying the regulation of mnp1 gene expression (Godfrey et al 1994). However, it would be useful to have systems that do not depend on the presence of a particular auxotrophic marker, and that are non-integrative.…”