Metabolic engineering of Catharanthus roseus hairy roots using an inducible promoter system
Hughes, Erik Heller
Doctor of Philosophy
Plant metabolic engineering is a developing field still in need of improved tools and an increased understanding of relevant pathways. This thesis addresses both those needs by testing a new tool for improved metabolic engineering studies and utilizing that tool for the exploration of monoterpenoid indole alkaloid biosynthetic pathways. Using GFP as a model protein in Catharanthus roseus hairy roots, we report that the glucocorticoid inducible promoter system is active and has a tightly controlled, reversible, and dosage-dependent response to dexamethasone. Furthermore, it provides an improved negative control useful for the study of genes affecting alkaloid synthesis. The most exhaustive transgenic studies reported here focus on the indole pathway. Expressing a feedback-resistant Arabidopsis anthranilate synthase alpha subunit results in dramatic increases in tryptophan and tryptamine yields. On induction, tryptophan increases from undetectable levels to 2.5 mg/g DW, while tryptamine increases from 25 mug/g DW to 267 mug/g DW. Additionally, a transient improvement in lochnericine yield indicates a possible increase in alkaloid flux countered by tight regulation of alkaloid levels. In lines transgenic for inducible tryptophan decarboxylase, serpentine specific yields increased by as much as 129% on induction. The reported studies on the indole pathway demonstrate successful methods to improve indole flux and show that increased indole flux can lead to improvements in certain alkaloids. Precursors from the complementing terpenoid pathway are also required for alkaloid synthesis. A feeding study utilizing an intermediate and a specific inhibitor of the nonmevalonate pathway validates the importance of this pathway for improved alkaloid yields and points to the potential success of upstream metabolic engineering efforts. In preliminary results, improved yields of certain alkaloids are reported for hairy root lines transgenic for 1-deoxy-D-xylulose-5-phosphate synthase and geraniol 10-hydroxylase, while an ORCA3 study highlights some potential problems with the use of transcriptional activators. In the last study, we focus on the engineering of a valuable alkaloid pathway and report a transgenic hairy root line overexpressing the full coding sequence of tabersonine 16-hydroxylase. On induction, the line produces 16-methoxytabersonine. Overall, a valuable new tool is introduced and subsequently used to manipulate the indole, terpenoid, and alkaloid pathways.
Agriculture; Plant Culture; Biochemistry; Chemical engineering