Functional expression of regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha spp.) in Escherichia coli and Saccharomyces cerevisiae

The oxygenation pattern of the essential oil monoterpenes of commercial min t (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In spearmi nt (M. spicata), C6-allylic hydroxylation leads to (-)-trans-carveol and th ence (-)-carvone, whereas in peppermint (M. x piperita), C3-allylic hydroxy lation leads to (-)-trans-isopiperitenol and ultimately (-)-menthol, cDNAs encoding the C6-hydroxylase and C3-hydroxylase from spearmint and peppermin t, respectively, were isolated by a combination of reverse genetic and homo logy-based cloning methods (S. Lupien, F. Karp, M. Wildung, and R. Croteau, Arch. Biochem. Biophys. 368, 181-192, 1999). Although both hydroxylase gen es were confirmed by functional expression using the baculovirus-Spodoptera system, too little protein was available by this approach to permit detail ed study of the structure-function relationships of these catalysts, especi ally the substrate binding determinants that underlie the regiochemistry an d stereochemistry of the reactions. Therefore, heterologous overexpression systems based on Escherichia coli and Saccharomyces cerevisiae were develop ed to produce several N-terminally modified versions of the recombinant hyd roxylases. Ancillary methods for the solubilization, purification, and reco nstitution (with recombinant spearmint cytochrome P450 reductase) of the li monene hydroxylases were also devised, with which substrate binding behavio r and precise regiochemistry and stereochemistry of product formation were determined, (C) 2000 Academic Press.