Genetic analysis of geraniol metabolism during fermentation

Abstract

Geraniol produced by grape is the main precursor of terpenols which play a key role in the floral aroma of white wines. We investigated the fate of geraniol during wine fermentation by Saccharomyces cerevisiae. The volatile compounds produced during fermentation of a medium enriched with geraniol were extracted by Stir-bar sorptive extraction and analysed by GC-MS. We were able to detect and quantify geranyl acetate but also citronellyl- and neryl-acetate. The presence of these compounds partly explains the disparition of geraniol. The amounts of terpenyl esters are strain dependant. We demonstrated both by gene overexpression and gene-deletion the involvement of ATF1 enzyme but not ATF2 in the acetylation of terpenols. The affinity of ATF1 enzyme for several terpenols and for isoamyl alcohol was compared. We also demonstrated that OYE2 is the enzyme involved in geraniol to citronellol reduction. Fermenting strain deleted from OYE2 gene produces far less citronellol than wild type strain. Moreover lab strain over-expressing OYE2 allows 87% geraniol to citronellol reduction in bioconversion experiment compared to about 50% conversion with control strain.

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

“…S. cerevisiae harbors two old yellow enzymes (OYE2 and OYE3) which catalyze the flavin-dependent, stereospecific reduction of α,β-unsaturated double bonds (Williams and Bruce, 2002). Previous studies concluded that OYE2 (but not OYE3) is involved in direct α,β-unsaturated monoterpenol reduction and is a target for increasing flux through the strictosidine pathway (Steyer et al, 2013). Other reports however, indicated that OYEs cannot reduce α,β-unsaturated alcohols, and that substrate reduction must proceed through activated α,β-unsaturated carbonyl intermediates (Brenna et al, 2012; Lonsdale and Reetz, 2015).…”

“…Thus, saturated shunt products represent a non-recoverable loss in carbon which severely limits overall iridoid yields. While several reports have described the reduction of the double bonds in monoterpene pathway intermediates by yeast (Gramatica et al, 1982; King and Richard Dickinson, 2000; Steyer et al, 2013), the exact mechanism has remained unresolved particularly with regard to crosstalk between exogenous biosynthetic enzymes (GOR and ISY) and endogenous yeast enzymes. However the low benchmark titers of 8-hydroxygeraniol 2 achieved to date (5.3 mg/L) (Campbell et al, 2016), in combination with the obscured mechanistic details of shunt product formation have significantly limited the reconstitution of nepetalactol 3 production and overall MIA pathway engineering in yeast.…”

De novo production of the plant-derived alkaloid strictosidine in yeast

“…Geraniol is metabolized by yeast primarily through esterification to form geranyl acetate and through reduction to form citronellol (37). Deletions of ATF1, encoding an alcohol acetyltransferase, and OYE2, encoding an NADPH oxidoreductase, are reported to attenuate these reactions (37). Thus, we deleted OYE2 and ATF1 in strain 1 to create strain 2.…”

Directed evolution and expression tuning of geraniol synthase for efficient geraniol production in <i>Escherichia coli</i>

“…The catalytic source of citronellol biosynthesis remains, as yet, unclear. In yeast, the enzyme (old yellow enzyme) responsible for the reduction of geraniol has been identified (Steyer et al, 2013). We deleted its homolog, nemA, but did not observe a decrease in citronellol production (Tashiro, M., unpublished data).…”