Date of Award
Doctor of Philosophy
Chemical and Biological Engineering
Jacqueline V. Shanks
The important anticancer pharmaceuticals, vinblastine and vincristine, are produced by Catharanthus roseus. Given their cytotoxicity, these valuable alkaloids are produced in very small quantities within the aerial parts of the plant. The high cost of isolating the drugs has led to research efforts to increase the alkaloid content of C. roseus cell cultures, tissue cultures, and seedlings. The metabolic engineering of C. roseus strives to overcome the strict regulation of the biosynthetic pathways.
Seedlings of C. roseus were elicited with methyl jasmonate (MeJA) to induce expression of octadecanoid–responsive Catharanthus AP2–domain 3 (ORCA3), a transcription regulator of several biosynthetic genes. ORCA3 exhibited increases up to 25–fold observed 0.5 h after MeJA treatment with the transcript levels of biosynthetic genes following with variable timing. The amounts of certain terpenoid indole alkaloid (TIA) metabolites, including the important vinblastine precursors, catharanthine and vindoline, were increased significantly.
Three hairy root cultures of C. roseus were investigated. The ASAB–1 line expressing a feedback–resistant anthranilate synthase (AS) α subunit from Arabidopsis under the control of a glucocorticoid–inducible promoter and an ASβ subunit from Arabidopsis under the control of the constitutive CaMV 35S promoter, the EHIDXS–4–1 line expressing 1–deoxy–D–xylulose 5–phosphate synthase (DXS) under the control of a glucocorticoid–inducible promoter, and the EHIT16H–34–1 line tabersonine 16–hydroxylase (T16H) under the control of a glucocorticoid–inducible promoter. These lines were used to investigate the regulatory nature of the biosynthetic network by quantifying the effect of light–adaptation, biosynthetic enzyme overexpression, and the combination of these two factors on the production of TIAs. Comprehensive metabolite profiling and a stoichiometric model were employed to reveal mechanisms of regulation. The results point towards controlling metabolite degradation as a potential focus for metabolic engineering efforts.
A proof of concept of a method for the introduction of 13C–labeling at the time of gene induction and preliminary results are presented. This method allows for the creation of metabolic flux maps of central carbon metabolism before and after the gene has been induced. The flux maps will reveal limitations in central carbon metabolism that affect the production potential of secondary metabolism.
The long term stability of a transgenic C. roseus hairy root line containing the inducible expression of a feedback–insensitive ASα is reported. After 5 years in liquid culture, the presence and inducible expression of the inserted AS gene was confirmed. This report also demonstrates that it may take as long as two years for the metabolite profile to stabilize.
Transgenic C. roseus hairy root lines were created that individually overexpress DXS and geraniol 10–hydroxylase (G10H) under the control of a glucocorticoid–inducible promoter. Double overexpression lines that overexpress DXS and ASα subunit or DXS and G10H with both genes under control of a glucocorticoid–inducible promoter were also created. The double overexpression lines displayed pertinent increases in TIA levels, surpassing the single overexpression lines.
The value of ultraviolet (UV) and mass spectra in identifying compounds in chromatographic methods is presented. The UV and mass spectra of important C. roseus secondary metabolites are included.
A method for the isolation of important C. roseus alkaloids is presented. A biomass extraction and analytical HPLC protocol was adapted for semi–preparative scale in order to obtain tabersonine, lochnericine, and hyrhammericine standards. Previously unidentified tabersonine–like compounds were also isolated for future identification.
Guy William Sander
Sander, Guy William, "Quantitative analysis of metabolic pathways in Catharanthus roseus hairy roots metabolically engineered for terpenoid indole alkaloid overproduction" (2009). Graduate Theses and Dissertations. 10820.