J. Microbiol. Biotechnol. J. Microbiol. Biotechnol. (2016), 26(12), 2076–2086 http://dx.doi.org/10.4014/jmb.1605.05090 Research Article jmb Review Improved NADPH Regeneration for Fungal Cytochrome P450 Monooxygenase by Co-Expressing Bacterial Glucose Dehydrogenase in Resting-Cell Biotransformation of Recombinant Yeast Hyunwoo Jeon 1† , Pradeepraj Durairaj 2,3† , Dowoo Lee 1 , Md Murshidul Ahsan 3 , and Hyungdon Yun 1 * Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea Korean Lichen Research Institute, Sunchon National University, Suncheon 57922, Republic of Korea School of Biotechnology, Yeungnam University, Gyeongsan 38541, Republic of Korea Introduction The Fungus kingdom, a large and diverse biological domain with 5.1 million fungal species, has extraordinary defense mechanic systems and unique metabolic flexibility facilitated by cytochrome P450 (CYP) monooxygenases [5, 7, 8, 27]. Although CYP enzymes are ubiquitous in all biological kingdoms, their divergence is manifold in the fungal kingdom and they play pivotal roles in various fungal metabolic processes, from housekeeping biochemical reactions, to detoxification of xenobiotics and production of metabolites critical for pathogenesis [7, 8, 20, 27]. Multiple fungal CYPs are involved in pathological virulence and are often part of biosynthetic gene clusters that process natural products like mycotoxins [16]. Among them, CYP53 enzymes (E.C. 1.14.13.12) are very essential in fungi as they play crucial roles in the detoxification and degradation of phenolic compounds (e.g., benzoate) produced by plants as a resistance mechanism against fungal infection [2]. Apparently, CYP53-mediated para-hydroxylation of benzoate Received: June 1, 2016 Revised: September 2, 2016 Accepted: September 5, 2016 First published online September 23, 2016 *Corresponding author Phone: +82-2-450-0496; Fax: +82-2-450-4769; E-mail: hyungdon@konkuk.ac.kr These authors contributed equally to this work. pISSN 1017-7825, eISSN 1738-8872 Copyright © 2016 by The Korean Society for Microbiology and Biotechnology 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°C. The “coupled- enzyme” system can certainly improve the overall performance of NADPH-dependent whole- cell biotransformations in a yeast system. Keywords: Cytochrome P450, benzoate hydroxylase, glucose dehydrogenase, heterologous expression, biotransformation, Saccharomyces cerevisiae