Stress as a means to enhance secondary metabolite productivity and to probe metabolic pathways

The objectives of this research were to study metabolic pathways in C. roseus hairy root cultures, and to enhance secondary metabolite productivity using fungal elicitation as the technique. The effects of age of inoculum were studied by adapting the cultures to three subculture cycle routines. The 2 week subculture cycle yielded the fastest, while the 4 week cycle yielded the lowest, specific growth rates. Specific yields of tabersonine decreased from day 21 to 35 while the total yields of horhammericine increased in all three subculture cycles. Lochnericine yields were highest in the 2 week cycle while serpentine yields were lowest. The effects of dosages and exposure times of specific elicitors on several compounds in the indole alkaloid pathway were studied. A 150% increase in tabersonine specific yield was observed upon addition of 72 units of pectinase. Levels of serpentine, tabersonine and lochnericine decreased transiently after addition of pectinase in time course studies. Jasmonic acid was found to be a unique elicitor leading to an enhancement in flux to several branches in the alkaloid pathway. Time course studies with jasmonic acid showed a transient increase in lochnericine and tabersonine levels and a continuous increase in levels of ajmalicine, serpentine and horhammericine. NMR spectroscopy was utilized as the tool to study primary metabolism of hairy roots non-invasively. \sp{31}P NMR spectroscopy studies indicated that vacuolar and cytoplasmic pH were maintained at 7.4 $\pm$ 0.06 and 5.25 $\pm$ 0.08 respectively. \sp{13}C NMR spectroscopy studies indicated activities of pentose phosphate pathways, non-photosynthetic CO\sb2 fixation and glucan synthesis pathways. Recycling of triose phosphate was evident from scrambling of label in glucans. In vivo \sp{31}P and \sp{13}C NMR spectroscopy was subsequently utilized to study the effects of elicitors on primary metabolism. A transient short-term decline in the cytoplasmic pH was observed upon addition of pectinase while a prolonged decrease in vacuolar pH was observed upon addition of jasmonic acid. Enhanced accumulation of glucans was detected upon addition of pectinase. A reduction in the levels of pyruvate and glutamine was observed, upon addition of jasmonic acid, indicating a decrease in flux to glycolysis or an increase in the drain on these pools