Fig. 1.

Engineering an orthogonal monoterpene biosynthetic pathway in yeast. The native yeast isoprenoid biosynthesis includes pathways responsible for the synthesis of sterols, dolichols, ubiquinone, as well as protein prenylation. Production of monoterpenoids in yeast has so far been based on utilizing the GPP synthesized as an intermediate in primary isoprenoid metabolism. This GPP-based non-orthogonal pathway (shown in gray) connects back to primary metabolism through the Erg20p-catalyzed conversion of GPP to FPP. Expression of SlNPPS1 in yeast cells enables the synthesis of the cis-isomer of GPP, NPP, which cannot be converted to FPP and links back to the biosynthesis of yeast isoprenoids. NPP can be utilized by engineered monoterpene synthases to produce different monoterpenes. The NPP-based pathway, depicted in green, is orthogonal to the yeast metabolism, because it branches out from the main pathway only at one point, following the synthesis of IPP and DMAPP. This branch point creates a metabolic valve, depicted here using the engineering symbol for a three-point valve, which can be regulated to direct fluxes to the desired products. This valve can be controlled dynamically to regulate fluxes between the two branches. We installed dynamic regulation by introducing the ergosterol-repressed promoter of the ERG1 gene upstream of the ERG20 gene. This valve diverts fluxes to the orthogonal branch when sterol levels increase. TPS terpene synthase, CYPs cytochrome P450 enzymes, ADHs alcohol dehydrogenases, GGPP geranylgeranyl diphosphate, P_ERG1 ERG1 promoter