Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae

• Published in: Metabolic engineering Vol. 18; pp. 25 - 35
• Main Authors: Gavira, Carole, Höfer, René, Lesot, Agnès, Lambert, Fanny, Zucca, Joseph, Werck-Reichhart, Danièle
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.07.2013; Elsevier
• Subjects: (+)-valencene; Arabidopsis; Biochemistry, Molecular Biology; Bioconversion; Biotransformation - genetics; Cellular Biology; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450; Life Sciences; Molecular biology; Nicotiana - enzymology; Nicotiana - genetics; Nootkatone; Oxidation; Plant Proteins - genetics; Plant Proteins - metabolism; Polycyclic Sesquiterpenes; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Sesquiterpenes - metabolism; Sesquiterpenes - pharmacology; (+)-valencene; Bioconversion; Oxidation; Nootkatone; Saccharomyces cerevisiae; Cytochrome P450
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2013.02.003

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 100mgL−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. •Tobacco CYP71D51v2 selectively converts (+)-valencene into β-nootkatol in vitro.•Yields of (+)-valencene bioconversion by CYP71D51v2-transformed yeast are low.•β-nootkatol and (+)-nootkatone are toxic for yeast at high concentration.•β-nootkatol accumulates in yeast endomembranes.•β-nootkatol is a ligand of CYP71D51v2 and inhibits (+)-valencene oxidation.