Improved NADPH Regeneration for Fungal Cytochrome P450 Monooxygenase by Co-Expressing Bacterial Glucose Dehydrogenase in Resting-Cell Biotransformation of Recombinant Yeast.
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 hydroxylaseFoCYP53A19and its homologous redox partner FoCPR fromFusarium oxysporumwere co-expressed with the BsGDH fromBacillus subtilisinSaccharomyces cerevisiaefor 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. TheFoCYP53A19+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°C. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.
Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea.
Hyunwoo
H
Jeon
Korean Lichen Research Institute, Sunchon National University, Suncheon 57922, Republic of Korea.|School of Biotechnology, Yeungnam University, Gyeongsan 38541, Republic of Korea.
Pradeepraj
P
Durairaj
Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea.
Dowoo
D
Lee
School of Biotechnology, Yeungnam University, Gyeongsan 38541, Republic of Korea.
Md Murshidul
MM
Ahsan
Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea.
Hyungdon
H
Yun
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D042843:Glucose 1-Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D001426:Bacterial Proteins; D005656:Fungal Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042843:Glucose 1-Dehydrogenase
Cytochrome P450; Saccharomyces cerevisiae; benzoate hydroxylase; biotransformation; glucose dehydrogenase; heterologous expression