Fungal cytochrome P450s participate in various physiological reactions, including the synthesis of internal cellular components, metabolic detoxification of xenobiotic compounds, and oxidative modification of natural products. Although functional analysis reports of fungal P450s continue to grow, there are still some difficulties as compared to prokaryotic P450s, because most of these fungal enzymes are transmembrane proteins. In this chapter, we will describe the methods for heterologous expression, in vivo analysis, enzyme preparation, and in vitro enzyme assays of the fungal P450 enzyme Trt6 and isomerase Trt14, which play important roles in the divergence of the biosynthetic pathway of terretonins, as a model for the functional analysis of fungal P450 enzymes.
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