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)
Mayfield
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MB
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, Portland 97291-1000.
Kishi
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Alic
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Gold
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D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D005800:Genes, Fungal / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010544:Peroxidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011523:Protoplasts / Q000382:microbiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014170:Transformation, Genetic
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D010544:Peroxidases; C051129:manganese peroxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases