Metabolic engineering of the terpenoid indole alkaloid pathway of Catharanthus roseus hairy roots
Peebles, Christie A. M.
Doctor of Philosophy
Catharanthus roseus is an important medicinal plant that produces a number of bioactive terpenoid indole alkaloids (TIAs). Specifically, vinblastine and vincristine and their semi-synthetic derivatives are important anticancer drugs used clinically. Low product yields have propelled researchers to pursue ways to increase TIA biosynthesis. Our group pursues this objective by developing tools to aid in the metabolic engineering of C. roseus hairy roots and by applying these tools to the study of pathway genes and regulators. Within this thesis, the long-term genetic and biochemical stability of 2 hairy root lines was confirmed after 4.5-5 years. This demonstrated the feasibility of using hairy roots in an industrial setting. The establishment of an ethanol inducible promoter provides another tool to study the expression of genes within C. roseus hairy roots. The expression of pathway genes and regulators was studied using a glucocorticoid-inducible promoter. Both a terpenoid precursor feeding study and the overexpression of 2 terpenoid genes (DXS and G10H) indicated that the engineering of the terpenoid pathway is an important target to increase the concentration of TIAs. Similarly, overexpression of one indole gene (ASalpha) and one terpenoid gene (DXS) also showed the importance of increasing the flux through each pathway. While single and double manipulations of pathway genes allow us to marginally increase the TIA concentration, targeting the expression of positive transcriptional regulators to simultaneously increase multiple genes could result in larger increases in TIA concentrations. The effect of expressing ORCA3, a jasmonate-responsive transcriptional regulator, along with jasmonic acid elicitation in hairy roots was tested. The effects of the manipulation were evaluated through the examination of both metabolite concentrations and mRNA transcripts over time. Interestingly jasmonic acid feeding alone caused the greatest increase in metabolites and mRNA transcripts. While ORCA3 expression was inducible, the potential positive effects seemed to be negated through the up-regulation of negative transcriptional regulators. These results show the importance of understanding transcriptional regulation in pathway manipulation. Overall this thesis advances our understanding of the metabolic engineering of the TIA pathway in C. roseus hairy roots.
Biomedical engineering; Chemical engineering