Identification of enzymes responsible for the reduction of geraniol to citronellol

Abstract

Abstract:The reduction of geraniol to citronellol is the first step for the synthesis of natural phytol in the production of tocopherols and natural vitamin K. Baker's yeast was used in the bioreduction described above as a whole-cell biocatalyst. However, the enzyme responsible for the reduction of geraniol to citronellol is not yet known. Four old yellow enzyme (OYE) genes were cloned from yeast and plants, and expressed in Escherichia coli for a high level of recombinant proteins. The recombinant protein displayed a catalytic activity of converting geraniol to citronellol as a sole product verified by GC-MS analyses. The recombinant OYE2 intact cells were found to show 3.7 and 1.9-fold higher activity than that of yeast cells and the recombinant crude extracts, respectively. Compared to the recombinant fusion enzyme, the entrokinase-cleaved enzyme displayed nearly identical activity for geraniol reduction. To our knowledge, this is the first enzyme identified to catalyze the formation of citronellol from geraniol by reducing the allylic alcohol double bond, which is normally known as inactivating group for the old yellow enzymes.

Genetic analysis of geraniol metabolism during fermentation

“…Considering the structural homologies between these two compounds and geranial and citronellal respectively, we hypothesized that transformation of geraniol to citronellol included aldehyde intermediates and that Oye2p was involved in geranial to citronellal reduction. Recently, Yuan et al (2011) have shown that crude extracts of Escherichia coli cells expressing Oye2p are able to reduce geraniol into citronellol but in vivo activity has not been evidenced. Therefore we tested Oye2p and the homologous Oye3p for their possible involvement in geraniol to citronellol transformation in fermentation experiments.…”

Biotransformation of hop derived compounds by Brettanomyces yeast strains

“…1) (3,7,8). Such a conversion has been correlated with expression of 'old yellow enzyme' (Oye2) by yeast, which is an oxidoreductase responsible for the transformation step (9,10). The citrusy flavour perceived in beers is caused by the synergistic effect of geraniol and citronellol (11).…”

Characterization of 12-Oxophytodienoic Acid Reductases from Rose-scented Geranium (Pelargonium graveolens)

“…Using the purified recombinant PgOPR1-3, the double-bond reducing activity was first assayed with geraniol as a substrate. However, GC-MS analyses of the reactions detected no products, indicating that geraniol is not a substrate for PgOPR1-3, contrary to the previous report that H. brasiliensis OPR and yeast OYE2, structurally related to PgOPRs, could accept geraniol as a substrate [12]. We thus next assayed enzyme activity of PgOPRs with citral as substrate.…”

“…This report provided a clue to the long-existing mystery of citronellol biosynthesis, but was rather surprising because OPR and OYE generally reduce double bonds adjacent to electronwithdrawing groups such as carbonyl groups [13]. Thus, geraniol, an allyl alcohol, did not seem to be a good substrate for OPR and OYE until Yuan's report was published [12]. However, if P. graveolens OPRs are also able to catalyze the geraniol to citronellol reduction, OPRs could be potential candidates for the citronellol biosynthetic enzyme in this plant.…”

“…However, the structure and function of geraniol reductase have long been unclear because geraniol reductase has neither been purified nor cloned to date. Recently, interesting results related to citronellol biosynthesis have been reported by Yuan et al [12]: They demonstrated that the purified recombinant Hevea brasiliensis 12-oxophytodienoic acid reductase (OPR) and Saccharomyces cerevisiae old yellow enzyme (OYE) 2 could catalyze the reduction of the double bond in geraniol to yield citronellol, as in the case of geraniol reductase reaction. This report provided a clue to the long-existing mystery of citronellol biosynthesis, but was rather surprising because OPR and OYE generally reduce double bonds adjacent to electronwithdrawing groups such as carbonyl groups [13].…”