KSEF RDE: Process engineering of fermentable yeast for high-value triterpene oil production

Triterpenes oils represent a special class of chemistry that play important biological roles in all living organisms, but they also have diverse industrial utility as vaccine adjuvants, emollients in cosmetics, as nutritional supplements and industrial lubricants. The traditional sources of these oils have been algal species whose growth is limited by current atmospheric conditions; or animal-based, specifically deep-water sharks, which have been overharvested for the high triterpene oil content in their livers. However, concerns over unreliable yields from algae sources, animal depletion and demands for cruelty-free natural products have re-focused efforts on obtaining these specialty oils from alternative, reliable and sustainable sources. The Chappell laboratory has recently captured the genetic blueprints for a wide array of triterpene oils from an ancient algal source and provided proof-of-principle evidence that the biosynthetic capacity for these unique oils can be engineered into microbial production platforms. The proposed aims for the current application is to advance our microbial production platform in 2 very specific ways. First, we will use new CRISPR-Cas9 methodology to insert the necessary genes encoding for this unique biosynthetic capacity into specific genetic loci of fermentable yeast lines, and evaluate these lines for their triterpene yields. Those lines exhibiting the highest biosynthetic capacity would then be diploidized in Specific Aim 2 and the resulting diploid lines qualified for triterpene oil production in process engineering experiments. The expected outcomes of the proposed activities would be yeast lines capable of producing 6 different triterpene oils poised for scale-up engineering and processing, as well as providing a greater understanding for building Synthetic Biology platforms for the production of chemicals with great, general utility.