Heterologous production of raspberry ketone in the wine yeast Saccharomyces cerevisiae via pathway engineering and synthetic enzyme fusion.

BACKGROUND: Raspberry ketone is the primary aroma compound found in raspberries and naturally derived raspberry ketone is a valuable flavoring agent. The economic incentives for the production of raspberry ketone, combined with the very poor yields from plant tissue, therefore make this compound an excellent target for heterologous production in synthetically engineered microbial strains. METHODS: A de novo pathway for the production of raspberry ketone was assembled using four heterologous genes, encoding phenylalanine/tyrosine ammonia lyase, cinnamate-4-hydroxlase, coumarate-CoA ligase and benzalacetone synthase, in an industrial strain of Saccharomyces cerevisiae. Synthetic protein fusions were also explored as a means of increasing yields of the final product. RESULTS: The highest raspberry ketone concentration achieved in minimal media exceeded 7.5 mg/L when strains were fed with 3 mM p-coumaric acid; or 2.8 mg/L for complete de novo synthesis, both of which utilized a coumarate-CoA ligase, benzalacetone synthase synthetic fusion protein that increased yields over fivefold compared to the native enzymes. In addition, this strain was shown to be able to produce significant amounts of raspberry ketone in wine, with a raspberry ketone titer of 3.5 mg/L achieved after aerobic fermentation of Chardonnay juice or 0.68 mg/L under anaerobic winemaking conditions. CONCLUSIONS: We have shown that it is possible to produce sensorially-relevant quantities of raspberry ketone in an industrial heterologous host. This paves the way for further pathway optimization to provide an economical alternative to raspberry ketone derived from plant sources.

Accumulation of guaiacol glycoconjugates in fruit, leaves and shoots of Vitis vinifera cv. Monastrell following foliar applications of guaiacol or oak extract to grapevines.

Previous studies have shown that volatile compounds present within a vineyard during the growing season can be absorbed by grapevines, assimilated within grapes, and then released during fermentation to influence the final aroma of wine. For example, the accumulation of volatile phenols in glycoconjugate forms following grapevine exposure to bushfire smoke, and their subsequent release during winemaking. This study investigated the accumulation of guaiacol glycoconjugates in the fruit, shoots and leaves of Monastrell grapevines following foliar applications (at veraison) of either an aqueous solution of guaiacol or an aqueous oak extract. Fruit, shoot and leaf samples were then collected at 3 time points between veraison and maturity, and analysed by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, to quantify guaiacol and its glycoconjugates, respectively. Guaiacol glycoconjugates were observed in fruit and leaves in particular, demonstrating glycosylation occurred after grapevine treatment; however, different glycoconjugate profiles were apparent.

Accumulation of Glycoconjugates of 3-Methyl-4-hydroxyoctanoic Acid in Fruits, Leaves, and Shoots of Vitis vinifera cv. Monastrell following Foliar Applications of Oak Extract or Oak Lactone.

Grapevines are capable of absorbing volatile compounds present in the vineyard during the growing season, and in some cases, volatiles have been found to accumulate in fruits or leaves in glycoconjugate forms, that is, with one or more sugar moieties attached. The presence of oak lactone in wine is usually attributable to oak maturation, but oak lactone has been detected in wines made with fruit from grapevines treated with oak extract or oak lactone. This study investigated the accumulation of glycoconjugates of 3-methyl-4-hydroxyoctanoic acid (i.e., the ring-opened form of oak lactone) in the fruits, leaves, and shoots of Monastrell grapevines following foliar application of either oak extract or oak lactone at approximately 7 days postveraison. Fruits, leaves, and shoots were collected at three different time points, including at maturity. The oak lactone content of fruit was determined by gas chromatography-mass spectrometry, with declining concentrations observed in fruit from grapevines treated with oak lactone with ripening. The concentrations of a ß-d-glucopyranoside of 3-methyl-4-hydroxyoctanoic acid in fruits, leaves, and shoots was determined by liquid chromatography-tandem mass spectrometry, with the highest oak lactone glucoside levels observed in leaves of grapevines treated with oak lactone. A glucose-glucose disaccharide was also tentatively identified. These results demonstrate both ring-opening and glycosylation of oak lactone occurred after experimental treatments were imposed.

Formation of Damascenone under both commercial and model fermentation conditions.

The fermentations, at a commercial winery, of six different grape musts encompassing the varieties Riesling, Chardonnay, Sauvignon blanc, Shiraz, Grenache, and Pinot noir were monitored for damascenone concentration. In every case, the concentration of damascenone increased during fermentation from low or undetectable levels to concentrations of several parts per billion. Further increases in damascenone concentration were observed during barrel aging of three of these wines. Two ketones, megastigma-4,6,7-triene-3,9-dione (4) and 3-hydroxymegastigma-4,6,7-trien-9-one (5), were synthesized and subjected to fermentation conditions using two yeasts, AWRI 796, and AWRI 1537. In the case of the former compound, 4, synthesis confirmed the original, tentative assignment of the structure and confirmed 4 as a natural product, isolated from honey. Both compounds, under the action of both yeasts, produced appreciable amounts of damascenone (1), with ketone 5 and AWRI 796 yeast yielding the highest concentration of 1.