Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae |
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| Institution: | 1. Institute of Plant Molecular Biology of CNRS UPR2357, University of Strasbourg, 28 rue Goethe, F-67083 Strasbourg Cedex, France;2. Laboratoire de Recherche en Biotechnologies, V. MANE & Fils, Bar-Sur-Loup, France;1. Departamento de Biotecnología, Universidad Autónoma Metropolitana, P.A. 55-535, 09340 Iztapalapa, México D.F., Mexico;2. Departamento de Química-Bioquímica, Instituto Tecnológico de Durango, Durango, Mexico;1. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, PR China;2. Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin 300350, PR China;3. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300350, PR China;1. State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, Laboratory of Molecular Biochemical Engineering, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong-chuan Road, Shanghai 200240, China;2. Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China;1. Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, USA;2. Disruptive & Sustainable Technologies for Agricultural Precision, Singapore-MIT Alliance for Research and Technology, Singapore;3. Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore;1. Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro, Tokyo 152-8551, Japan;2. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan;3. Department of Applied Biological Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan;4. Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan |
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| Abstract: | Natural nootkatone is a high value ingredient for the flavor and fragrance industry because of its grapefruit flavor/odor, low sensorial threshold and low availability. Valencene conversion into nootkatol and nootkatone is known to be catalyzed by cytochrome P450 enzymes from both prokaryotic and eukaryotic organisms, but so far development of a viable bioconversion process using either native microorganisms or recombinant enzymes was not successful. Using an in silico gene-mining approach, we selected 4 potential candidate P450 enzymes from higher plants and identified two of them that selectively converted (+)-valencene into β-nootkatol with high efficiency when tested using recombinant yeast microsomes in vitro. Recombinant yeast expressing CYP71D51v2 from tobacco and a P450 reductase from arabidopsis was used for optimization of a bioconversion process. Bioconversion assays led to production of β-nootkatol and nootkatone, but with low yields that decreased upon increase of the substrate concentration. The reasons for this low bioconversion efficiency were further investigated and several factors potentially hampering industry-compatible valencene bioconversion were identified. One is the toxicity of the products for yeast at concentrations exceeding 100 mg L−1. The second is the accumulation of β-nootkatol in yeast endomembranes. The third is the inhibition of the CYP71D51v2 hydroxylation reaction by the products. Furthermore, we observed that the formation of nootkatone from β-nootkatol is not P450-dependent but catalyzed by a yeast component. Based on these data, we propose new strategies for implementation of a viable P450-based bioconversion process. |
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| Keywords: | (+)-valencene Nootkatone Oxidation Cytochrome P450 Bioconversion |
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