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The CYPome of the model xenobiotic-biotransforming fungus Cunninghamella elegans.

ABSTRACT: The fungus Cunninghamella elegans is recognised as a microbial model of mammalian drug metabolism owing to its ability to catabolise xenobiotic compounds in an analogous fashion to animals. Its ability to produce phase I (oxidative) metabolites of drugs is associated with cytochrome P450 (CYP) activity; however, almost nothing is known about these enzymes in the fungus. In this paper we report the in silico analysis of the genome sequence of C. elegans B9769, which contains 32 genes putatively coding for CYPs. Based on their predicted amino acid sequences these were classified as belonging to CYP509, 5203, 5208, 5313, 5210, 61 and 51 families. Reverse transcription-quantitative PCR revealed that the gene coding for CYP5313D1 was significantly upregulated when C. elegans DSM1908 was cultivated in sabouraud dextrose in contrast to its expression in cells grown in Roswell Park Memorial Institute medium. This corresponded to the fungus' xenobiotic biotransformation ability when grown in the two media. Heterologous expression of cyp5313D1 in Pichia pastoris resulted in a recombinant strain that biotransformed flurbiprofen to 4'-hydroxyflurbiprofen, the same metabolite generated by C. elegans cultures. This is the first report of a xenobiotic-biotransforming CYP from this biotechnologically important fungus.

SUBMITTER: Palmer-Brown W

PROVIDER: S-EPMC6592952 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Dihydroartemisinin (DHA) is a sesquiterpene endoperoxide with prominent antimalarial efficacy, which was discovered by Professor Youyou Tu through the reduction of artemisinin in the 1970s. It is always a challenging work for scientists to investigate the metabolites of DHA in the red blood cells due to the complicated matrix background. As a bottleneck, the investigation of metabolites, especially exploring the pharmacodynamic material in the red blood cell, is necessary and significant for metabolism research of antimalarial agent. Recently, microbial transformation provides a green and economical means for mimicking mammal metabolism and synthesis active metabolites, based on which is one efficient route for drug discovery. In this study, a strain from Cunninghamella was employed as an efficient tool to explore active metabolites of DHA in erythrocyte. Microbial transformation products of DHA by Cunninghamella elegans CICC 40250 were detected and analyzed by ultra-performance liquid chromatography (UPLC)-electrospray ionization (ESI)-quadrupole time-of-flight (Q-TOF)-mass spectrometry (MSE), and the main products were isolated and identified. The antimalarial activity of the isolated products was also screened in vitro. Totally, nine products were discovered through UPLC-ESI-QTOF-MSE, and three main products with novel chemical structures were isolated for the first time, which were also detected in red blood cells as the metabolites of DHA. After evaluation, 7β-hydroxydihydroartemisinin (M1) exhibited a good antimalarial activity with an IC50 value of 133 nM against Plasmodium falciparum (Pf.) 3D7. The structure and stereo-configuration of novel compound M1 were validated via X-ray single crystal diffraction. Microbial transformation was firstly employed as the appropriate model for metabolic simulation in erythrocyte of DHA. Three novel metabolites in erythrocyte were obtained for the first time through our microbial model, and one of which was found to show moderate antimalarial activity. This work provided a new research foundation for antimalarial drug discovery.

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The CYPome of the model xenobiotic-biotransforming fungus Cunninghamella elegans.

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