Wednesday, August 13, 2008 - 10:00 AM

Using Saccharomyces cerevisiae for production of terpenoid compounds for use as fragrances and flavorings

Grace Park, Bryan Greenhagen, Kevin Nowaczyk, Richard Burlingame, and Bryan Julien. Allylix, 1500 Bull Lea Rd., Lexington, KY 40511

Many compounds used as fragrances and flavorings belong to the class of plant molecules called terpenoids. The availability of these compounds can vary due to environmental conditions, and their chemical synthesis can be expensive. To provide a cost effective and sustainable source of these compounds, an engineered Saccharomyces cerevisiae has been developed. Nootkatone, the molecule that gives grapefruit its distinctive smell and taste, is a member of this family. It is used currently as a flavoring for beverages but has other potential uses if produced more economically. Nootkatone is synthesized using the citrus valencene synthase (CVS) enzyme to convert FPP (farnesyl pyrophosphate) to valencene. Valencene is then oxidized, either biologically or chemically, to produce nootkatone. Expression of the native CVS enzyme in S. cerevisiae engineered to overproduce FPP results in low-level production of valencene. Using a high throughput screen, mutants with increased valencene production have been identified. Combining mutations has resulted in a highly mutated valencene synthase with a significant increase in productivity. In addition to CVS enzyme improvements, engineering of the host strain was done to increase overall productivity. The original production strain required the addition of ergosterol due to the deletion of ERG9, the squalene synthase gene, which increased the FPP pool. However, the ability of this strain to grow to high cell densities was hindered. To overcome the growth defect, we have engineered ERG9 so that the strain no longer requires sterols for growth. This strain grows to a higher cell density, and has increased productivity.