Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium

• Published in: Applied and Environmental Microbiology 1994-12, Vol.60 (12), p.4303-4309
• Main Authors: MAYFIELD, M. B, KISHI, K, ALIC, M, GOLD, M. H
• Format: Article
• Language: English
• Subjects: actividad enzimatica; activite enzymatique; adn recombinado; adn recombine; Bacteria; Bacteriology; Base Sequence; Basidiomycota - enzymology; Basidiomycota - genetics; Basidiomycota - metabolism; Biochemistry; Biological and medical sciences; Biotechnology; Cloning, Molecular; enzymic activity; Fundamental and applied biological sciences. Psychology; gene; Gene Expression Regulation, Fungal - genetics; Genes; Genes, Fungal - genetics; Genetic engineering; Genetic technics; genetic transformation; Genetic Vectors - genetics; gliceraldehido 3 fosf deshidrog; glyceraldehyde 3 phosphate dehydrog; glyceraldehyde 3 phosphate deshydro; Glyceraldehyde-3-Phosphate Dehydrogenases - genetics; Hydrogen-Ion Concentration; Isoenzymes - biosynthesis; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kinetics; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; Molecular Weight; peroxidasas; peroxidases; Peroxidases - biosynthesis; Peroxidases - chemistry; Peroxidases - genetics; Peroxidases - isolation & purification; Peroxidases - metabolism; peroxydase; Phanerochaete chrysosporium; Promoter Regions, Genetic - genetics; Protoplasts - microbiology; recombinant dna; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; transformacion genetica; transformation genetique; Transformation, Genetic
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.60.12.4303-4309.1994

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 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 site-directed mutants of Mn peroxidase.