"Hairy" roots hold the potential for economically feasible biotechnological routes to the controlled biosynthesis of complex, plant-derived, 'natural' molecules. A novel transgenic root system of the tropical plant Catharanthus roseus was established and analyzed for the synthesis of indole alkaloids, including the valuable anti-cancer drugs vinblastine and vincristine. A structured approach to developing the biosynthetic potential of hairy roots is presented: transformation, screening, selection, optimization of culture protocols, and product enhancement.
Five hairy root clones with unoptimized doubling times of 3-4 days and vindoline output of 0.005-0.07% dry weight, were screened from 150 transformants. Hairy root morphology likely under the control of rol genes transferred from the Agrobacterium plasmid, was identified as a key determinant of fitness in liquid culture and a target for transgenic design for large-scale bioreactor environments.
The hairy root inoculum was optimized and standardized to facilitate the assessment of culture performance under diverse environmental treatments and in process scale-up. The length of the root tip has a dominant effect on growth, uninfluenced by clonal variability. The optimum inoculum is comprised of 5 root tips, each 35-40 mm long, in 50 mL media.
Long-term dose-response and transient studies examined heterotrophic and photoheterotrophic carbon regimes. These studies are unique in the metabolic adaptation of cultures, and examined the putatively antagonistic kinetics of nutrient utilisation and secondary metabolite accumulation. The activities of the cathenamine and bisindole alkaloid pathways responded, respectively to high and moderate sucrose concentrations. The cultures were nitrogen limited with 2-4% sucrose in B5/2 salt. Organic acids were excreted in the presence of excess sugars. The ordered assimilation of macronutrients--ammonium, phosphate and nitrate--corroborated by changes of extracellular pH, have important implications on fed-batch strategies. Tabersonine accumulation was growth-associated, while serpentine accumulated in a non-growth manner. Ajmalicine, catharanthine, vindoline, and compounds tentatively identified as vinblastine and vincristine, accumulated optimally in the late-exponential or early-stationary phase. Photoheterotrophic conditions incremented peak biomass by 60-300%, doubling times by 60%, and vindoline levels by an order of magnitude, likely due to the anapleurotic activity of PEPCase and the light induction of nitrate reductase and vindoline synthesis.