Ganoderma lucidum, (Leyss. ex Fr.) Karst (Fr.) Karst

Yang, Chengshuai, Li, Weichao, Li, Chen, Zhou, Zhihua, Xiao, Youli & Yan, Xing, 2018, Metabolism of ganoderic acids by a Ganoderma lucidum cytochrome P 450 and the 3 - keto sterol reductase ERG 27 from yeast, Phytochemistry 155, pp. 83-92 : 87

2.5. In vitro enzymatic activity confirmed the function of GLCPR, an NADPH-CYP reductase from G. lucidum

In general, a CYP requires an NADPH-CYP reductase for its function. In S. cerevisiae , the NADPH-dependent CYP reductase, NCP1, was characterized as a working partner of endogenous or heterogeneous CYPs ( Hirosue et al., 2011; Ide et al., 2012; Venkateswarlu et al., 1997). An ORF annotated as an NADPH-dependent CYP reductase, which shares a 36.62% amino acid sequence similarity with NCP1, was cloned from G. lucidum cDNA and named GLCPR. GLCPR was then introduced into a yeast chassis to replace the full-length ORF of NCP1 and the recombinant GLCPR113-3C strain was able to grow normally in YPD medium without the addition of ergosterol. It was demonstrated that the heterogenous GLCPR functioned in cooperation with endogenous CYPs in yeast to produce ergosterol, which is essential for yeast growth. Moreover, the pESC-dTrp-GLCPR-CYP512U6 expression vector containing the CYP512U6 and GLCPR ORFs under the control of the GAL1 and GAL10 promoters respectively, was constructed in GLCPR113-3C, and the resulting strain ZYGL02 catalyzed the conversion of ganoderic acid DM to 2 ( Fig. 5 and Table 1 ), indicating that the GLCPR functions in cooperation with CYPs such as CYP512U6, in G. lucidum .

Figures

Fig. 5. Functional identification of GLCPR. Trace a shows the authentic ganoderic acid DM; Traces b, c, d and e show the analyses of products from the boiled microsomal fractions of ZYGL00, the microsomal fractions of ZYGL00, the microsomal fractions of ZYGL02, and the boiled microsomal fractions of ZYGL02 with ganoderic acid DM.