REGULATION AND HETEROLOGOUS EXPRESSION OF P450 ENZYME SYSTEM COMPONENTS OF THE WHITE ROT FUNGUS PHANEROCHAETE CHRYSOSPORIUM

Abstract

Phanerochaete chrysosporium is widely used as a model organism to understand the physiology, enzymology, and genetics of lignin degradation by white rot fungi and is known for its ability to metabolize and detoxify a wide range of environmental chemicals. Our pre-genomic efforts and the recent whole genome sequencing by the Joint Genome Institute of the US-DOE have revealed that this fungus carries a well developed P450 enzyme system, consisting of multiple P450 monooxygenases and a common P450 oxidoreductase. The entire P450ome of this organism comprises of ~150 cytochrome P450 monooxygenases, mostly arranged in gene clusters and classifiable into multigene families. Except for the structurally and functionally conserved fungal P450 families such as CYP51, CYP61, and CYP53, other P450 enzymes in this organism have largely unknown function and will require functional characterization. These new P450 enzymes may likely have roles in biodegradation activity and physiology of this ligninolytic fungus. Our pre- and post-genomic efforts to understand the functional role of P450 enzyme systems in P. chrysosporium have focused on the regulation of expression of the first identified family of P450 enzymes, the CYP63 family, and genome-wide regulation of the other P450 families using a custom-designed P450 microarray. The genomically-linked CYP63 member P450s were found to be differentially regulated under varying physiological and/or biodegradation conditions. Results on the heterologous expression of this family of monooxygenases in different prokaryotic and eukaryotic expression systems are presented and the inherent problems associated with the expression of these membrane proteins are discussed. Further, we report the expression and purification of the white rot fungal cytochrome P450 oxidoreductase (POR), the electron transfer component of its P450 enzyme system, required for P450 catalysis. The reported studies have uncovered the hitherto unknown regulatory aspects of the P450 enzyme system in P. chrysosporium and generated useful expression tools and knowledgebase to pursue further studies on functional analysis of the P450 contingent in this model white rot fungus.

Fig. 1

Sequence alignment of the native sequence (pc-1) and the codon-optimized sequence (pc-1-syn) of CYP63A1 cDNA of P. chrysosporium. MegAlign 5.05 and GeneDoc Version 2.6.002 softwares were used for the alignment. Altered bases are not highlighted.

Fig. 2

Heterologous expression of the white rot fungal P450 monooxygenases PC-1 and PC-3 in E. coli. A: Left panel, Effect of temperature on PC-1 expression. Right panel, Expression of PC-1 in E. coli at 37 °C. 1 – uninduced inclusion bodies, 2 – induced inclusion bodies, 3 – vector control, 4 – induced soluble fraction, 5 - uninduced soluble fraction. B: Left panel, Effect of temperature on PC-3 expression. Right panel, Expression of PC-3 in E. coli at 37 °C. 1 – uninduced soluble fraction, 2 – induced soluble fraction, 3 – vector control.

Fig. 3

Heterologous expression of the white rot fungal P450 monooxygenase PC-1 in eukaryotic expression systems. A. PC-1 expression in Saccharomyces cerevisiae. Yeast cultures grown in SCD-ura and induced with 2% galactose for varying incubation times (0, 4, 8, 12, 16, 20, and 24 h) were harvested and the expressed protein was detected using anti-his antibody. B. PC-1 expression in Baculoviral cell line. Sf9 cells expressing PC-1 protein were harvested after 24, 48, 72, and 96 h post addition of hemin and the expressed protein was detected using anti-PC-1 antibody.

Fig. 4

Heterologous expression of the white rot fungal P450 oxidoreductase (POR) in E. coli and its purification. A. Total protein extract was passed through Ni-NTA column followed by washing and elution with increasing concentrations of imidazole. An equal volume was loaded on a 10% SDS-PAGE gel followed by silver staining of the gel. B. Western blot analysis using anti-POR antibody. M, Marker; F, Flowthrough; W, Washing; E, Elutions.