Application of site-specific endonuclease technology to rapid expansion of product portfolio in engineered yeast

Terpenes constitute the largest group of plant natural products, and have long been used as fragrances and flavors, pharmaceutical agents and insecticides. At Amyris we engineer yeast to produce terpene-based renewable chemicals and biofuels. Our current portfolio of commercial products includes the anti-malarial drug artemisinin, renewable diesel, and squalane as a renewable emollient for cosmetics. We are actively expanding our portfolio of products, and developing technologies that expedite the development of new products. Here we describe the use of endonucleases-initiated site-specific homologous recombination to convert our farnesene producing yeast strains into strains producing a wide variety of terpene-based chemical products. Site-specific endonucleases cause DNA double-strand breaks that facilitate the incorporation of exogenous DNA at targeted sites through homologous recombination. Here we demonstrate simultaneous recombination at up to four endonuclease cut sites in a single transformation, successfully converting high-level farnesene producing strains into strains producing another terpene. This technology enables us to leverage the farnesene strain engineering we have performed to-date, and will greatly accelerate the development of new terpene products in the future.