| This dissertation describes studies of isoprenoid biosynthesis in the phytoflagellate Euglena gracilis. Elucidation of a novel isoprenoid pathway, the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, in addition to the generally known mevalonate (MVA) pathway, led to extensive exploration of the distribution of both pathways in a variety of organisms. To date, photosynthetic eukaryotes have been known to possess both pathways in separate compartments, such as the cytosol and chloroplasts. They are able to utilize the MVA pathway to produce cytosolic isoprenoids such as sterols and the MEP pathway to synthesize phytol and carotenoids in the chloroplasts. Euglena gracilis, however, was regarded as the only photosynthetic eukaryote in which the MEP pathway was not detected.; In this dissertation, isoprenoid biosynthesis in E. gracilis strain Z was investigated using incubation experiments with the labeled precursors [1-13C]-glucose, [5-2H 2]-deoxy-D-xylulose, (U-13C6]-glucose, [1,2- 13C2]-acetate, [methyl-13C]-methionine, [2-13C]-leucine, [1-13C]-butyric acid, [1,2,3,4- 13C4]-acetoacetyl-N-acetylcysteamine, [1,2,3,4-13C4]-dimethylacrylic acid, 3-[methyl- 2H3]-3-butenoic acid, 3-[methyl-2H 3]-3-butenoyl-N-acetylcysteamine, [2-13C]-malonic acid, [1-13C-2-2H3]-acetate, and [2-13C2-2H3]-acetate. Several isoprenoid compounds were analyzed, such as carotenoids (beta-carotene and diadinoxanthin), phytol, alpha-tocopherol and ergosterol. These experiments provided new findings in contrast to previous results, which are: (1) E. gracilis does indeed possess the MEP pathway and utilizes both the MVA and the MEP pathways to produce isoprenoid compounds, especially carotenoids, and (2) phytol appears to be synthesized by a polyketide-type pathway, rather than an isoprenoid pathway.; Therefore, the existence of the MEP pathway to isoprenoids in E. gracilis is first described in this dissertation. Also strong evidence is provided that phytol biosynthesis in E. gracilis is not via an isoprenoid pathway, but rather by a polyketide-type pathway. Further studies will be necessary to explore the details of this unprecedented route to phytol. |
