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Production of Farnesol and Geranylgeraniol
by Strains of Saccharomyces cerevisiae



Isoprenoids represent the most diverse group of naturally occurring compounds, and are derived from the 5-carbon molecule isopentenyl pyrophosphate (IPP). Two distinct biosynthetic pathways have been identified for the synthesis of IPP. One pathway found in animals, plants, and some bacteria uses acetyl CoA to form mevalonate, which is further metabolized to IPP. The other more recently discovered pathway is also found in plants, as well as many bacteria, and utilizes glyceraldehyde-3-phosphate and pyruvate to form deoxyxylulose-5-phosphate, which is further metabolized to IPP. IPP monomers are then condensed by a number of enzymes with different chain length specificities to form isoprenoids of varying lengths. These isoprenoids can be further modified in a number of ways to form a vast array of compounds.

The yeast Saccharomyces cerevisiae synthesizes isoprenoids via the mevalonate dependent pathway, and relies on this pathway to supply precursors for the synthesis of ergosterol, ubiqinone, heme A, and dolichol, as well as for protein prenylation. The isoprenoid pathway is completely defined in this organism, and all of the genes for isoprenoid pathway enzymes have been identified and made available for improving organisms. Farnesyl diphosphate is a branch point intermediate, contributing to the synthesis of the compounds listed above; however, the majority of the carbon moving through this pathway is directed toward the biosynthesis of ergosterol. The first step leading from farnesyl diphosphate to ergosterol is carried out by the enzyme squalene synthase. In S. cerevisiae, this enzyme is encoded by the ERG9 gene. Condensation of farnesyl diphosphate with IPP leads to the formation of geranylgeranyl diphosphate (GGPP), which is used to prenylate specific proteins. This reaction is carried out by the enzyme GGPP synthase, encoded by the BTS1 gene in S. cerevisiae.

Normally, the isoprenoid pathway is tightly regulated at the level of HMG CoA reductase. In S. cerevisiae, two isoforms of this enzyme exist, coded by the HMG1 and HMG2 genes. These genes and enzymes are regulated at multiple levels including transcription, translation, and protein degradation. However, the isoprenoid pathway can be modified for high flux, as demonstrated by the isolation of yeast strains capable of accumulating as much as 15-20% of their dry cell weight as squalene, ergosterol, and other sterols (Saunders et al., U.S. Patent 5,460,949; 1995). The two yeast HMG CoA reductases are both composed of an amino-terminal membrane spanning domain and a carboxy-terminal catalytic domain. R. Hampton (Department of Biology, U.C. San Diego) constructed plasmids capable of expressing in yeast the catalytic domains of both HMG1 and HMG2 (referred to here as HMG1cat and HMG2cat). These genetic constructs remove the regulation normally imposed upon this step of the pathway. Elevated expression of the deregulated HMG1cat gene in yeast has been shown to increase carbon flux into the isoprenoid pathway, as measured by an increase in the accumulation squalene and sterols (Saunders et al., U.S. Patent 5,460,949; 1995; Donald et al., Applied and Environmental Microbiology, 63:3341-3344; 1997).

Isoprenoids are components of a variety of interesting molecules such as carotenoids, ubiquinones, steroids, prenylated proteins, and certain vitamins and pharmaceuticals. Bio-Technical Resources is interested in developing fermentation processes for the biological production of isoprenoids that can be used as intermediates in the synthesis of commercially valuable chemicals, including vitamin E (Millis et al., U.S. Patent 6,242,227; 2001). We chose to develop this system in S. cerevisiae, taking advantage of the tools and knowledge of this industrially proven organism to manipulate the isoprenoid pathway for the production of both farnesol and geranylgeraniol.