Ene-Reductase Catalyzed Regio- and Stereoselective 1,4-Mono-Reduction of Pseudoionone to Geranylacetone

The regio- and stereoselective mono-reduction of a particular C=C bond of conjugated C=C double bonds is a very challenging task. Here the regio- and stereoselective 1,4-reduction of pseudoionone, an alpha,beta,gamma,delta-bisunsaturated ketone, was demonstrated to give geranylacetone, an industrially relevant molecule. OYE1 from Saccharornyces postorianus was identified as the most suitable biocatalyst for this reaction. Elevated substrate concentrations of up to 200 mM were tolerated allowing still to reach excellent conversions (>99% and 80% for 100 or 200 mM pseudoionone concentration, respectively). Interestingly, the organic cosolvent often required for substrate solubilization in aqueous buffer can be avoided for pseudoionone when using permeabilized E. coli cells containing the overexpressed enzyme instead of purified enzyme, reaching still >99% conversion at 100 mM (19.2 g/L) substrate concentration. Performing this reaction at a 0.5 g scale allowed to run the reaction to completion (> 99%) and pure product was isolated with 80% yield. Additionally, the bis-unsaturated ketone 6-methyl-3,5-heptadien-2-one was transformed under similar conditions giving the floral compound sulcatone with excellent conversion (97%) and 77% isolated yield. Finally, the stereoselective reduction of the (E,E)- over the (E,Z)-pseudoion-one isomer was enabled by the ene-reductase from Zyrnornonas mobilis (NCR). Thus, both (E)-geranylacetone and (E,Z)-pseudoionone were obtained with isomeric excess above 60%.

Automated summary

The study demonstrates a regio- and stereoselective mono-reduction method for pseudoionone using OYE1 from Saccharornyces postorianus, achieving excellent conversion at high substrate concentrations. Furthermore, utilizing E. coli cells containing overexpressed enzyme instead of purified enzyme allows for efficient conversion without the need for organic cosolvent. In addition, the ene-reductase from Zyrnornonas mobilis enables stereoselective reduction of the (E,Z)-pseudoion-one isomer, providing both (E)-geranylacetone and (E,Z)-pseudoionone with high isomeric excess.