Macrocyclic diterpenoids produced in plants from the Euphorbiaceae family show a multiplicity of biological activities and provide new leads for the development of pharmaceuticals against a wide range of medical conditions. Biosynthesis of such compounds in microbial hosts promises to overcome the limitations of conventional production systems, such as chemical synthesis and extraction from plants. This PhD thesis reports on the investigation of the biosynthetic pathway to ingenol-]-angelate, a diterpenoid ester approved for the treatment of actinic keratosis, a pre-cancerous skin lesion. The yeast Saccharomyces cerevisiae was engineered to develop a biological chassis not only for elucidation of parts of the biosynthetic pathway towards ingenol-]-angelate, but also for the generation of a potential production host for valuable diterpenoid compounds. Engineering of the yeast native mevalonate pathway showed significant improvements in production of the diterpene hydrocarbon casbene, the first committed precursor within the pathway to ingenol. Dynamic control of the ERGJK and ERGL genes by means of ergosterol- and glucose- sensitive promoters, together with simultaneous expression of a prenyltransferase domain from Phomopsis amygdali and a casbene synthase from Ricinus communis, allowed for production of casbene in yields of POQ. mg/L. The casbene modifying enzymes CYP\PDXX , CYP\N`AN\ and ADHP from Euphorbia lathyris L. were functionally characterized in yeast and showed to be responsible for specific hydroxylations of the casbene backbone, followed by an intramolecular carboncarbon ring closure leading to synthesis of jolkinol C - a potential key intermediate in ingenol biosynthesis. Further optimization of the culturing conditions of yeasts expressing CYP\PDXX , CYP\N`AN\ and ADHP allowed for production of a compound with the same mass and retention time as ingenol.
Finally, biosynthesis of angelyl-CoA, the CoA-activated form of angelic acid needed for angelyl-acylation of ingenol, was achieved. Genes from the ssf cluster of Streptomyces sp. SFN \ were expressed in yeast and led, upon propionic acid supplementation and further modifications in the pathway, to maximally `.X mg/L of angelyl-CoA. Moreover, feeding of angelic acid to yeast strains expressing acyl-CoA ligases from plant species allowed for a production of angelyl-CoA in the range of XO mg/L. The results of this thesis show the potential of employing Saccharomyces cerevisiae for pathway elucidation and biotechnological production of pharmaceutically active plant diterpenoids.