• The Korean Journal of Nuclear Medicine
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• v.38 no.4
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• pp.325-329
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• 2004

Purpose: Usefulness of mouse liver S9 fraction was evaluated for the measurement of the metabolites in the in vitro metabolism study of $^{18}F$-labeled radiotracers. Materials and Methods: Mouse liver S9 fraction was isolated at au early step in the course of microsome preparation. The in vitro metabolism studies were tarried out by incubating a mixture containing the radiotracer, S9 fraction and NADPH at $37^{\ciirc}C$, and an aliquot of the mixture was analyzed at the indicated time points by radio-TLC. Metabolic defluorination was further confirmed by the incubation with calcium phosphate, a bone mimic. Results: The radiotracer $[^{18}F]1$ underwent metabolic defluorination within 15 min, which was consistent with the results of the in vivo method and the in vitro method using microsome. Radiotracer $[^{18}F]2$ was metabolized to three metabolites including $4-[^{18}F]fluorobenzoic$ acid within 60 min. It is likely that the one of these metabolites at the origin of radio-TLC was identical with the one that obtained from the in vivo and in vitro (microsome) method. Compared with the in vitro method using microsome, the method using S9 fraction gave a similar pattern of the metabolites but with a different ratio, which can be explained by the presence of cytosol in the S9 fraction. Conclusion: These results suggest that the findings of the in vitro metabolism studies using S9 fraction can reflect the in vivo metabolism of novel radiotracers in the liver. Moreover, this method can be used as a tool to determine metabolic defluorination along with calcium phosphate absorption method.

• Journal of the Korean Wood Science and Technology
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• v.44 no.2
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• pp.253-263
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• 2016

Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Ten additional species of white rot fungi were inoculated in medium containing nutrients such as $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ at $28^{\circ}C$ for 25 days. After 10 days of incubation, eudesmane-type sesquiterpenes, ${\beta}$-eudesmane and ${\beta}$-eudesmol, were only synthesized during the growth phase of P. brumalis. Experiments excluding one nutrient at a time were conducted to determine the effects of inorganic nutrients on sesquiterpene biosynthesis. In conclusion, GC-MS analysis showed that biosynthesis of sesquiterpenes was differentially regulated by inorganic nutrients such as $MgSO_4$, $C_4H_{12}N_2O_6$, and $KH_2PO_4$. We found $MgSO_4$ supplementation to be vital for eudesmane-type sesquiterpene biosynthesis in P. brumalis; nitrogen ($C_4H_{12}N_2O_6$) and phosphate ($KH_2PO_4$) inhibited the synthesis of P. brumalis metabolites. Magnesium is a known cofactor of sesquiterpene synthase, which promotes ${\beta}$-eudesmol synthesis. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2076-2086
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• 2016

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at $30^{\circ}C$. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.