Metabolic studies of Catharanthus roseus hairy root cultures by phosphorus-31 and carbon-13 nuclear magnetic resonance spectroscopy

[摘要] The regulation of primary metabolism and induction of secondary metabolism in Catharanthus roseus hairy root cultures were studied using noninvasive in vivo NMR spectroscopy. A micro-perfusion system was first developed and characterized for the in vivo NMR metabolic studies which include both short-term bioassays and long-term micro-reactor experiments. In vivo $sp{31}$P NMR experiments indicated that hairy roots stored P$sb{i}$ in the vacuoles. Cytoplasmic and vacuolar pH of C. roseus hairy roots were regulated at 7.40 $pm$ 0.08 and 5.45 $pm$ 0.12 respectively with initial medium pH at 5.7, 6.5 or 7.3. A more acidic vacuolar pH and abnormal root morphology were observed at external pH of 4.2. Utilization of inorganic phosphates was unaffected by this morphological variation. Significantly higher specific yields of vindoline and catharanthine were found in the cultures initiated at pH 4.2. In vivo $sp{13}$C NMR data indicated active pentose phosphate pathway in the metabolism of C. roseus hairy root cultures. Transient profiles of glucan resonances has led to a dynamic model for glucan synthesis. Under hypoxic conditions, C. roseus hairy roots accumulated ethanol, malate and $gamma$-aminobutyric acid, and the cytoplasmic pH dropped. Glycerol was synthesized uniquely in C. roseus hairy roots perfused with medium containing glucose. Variations in shear force or in gas composition are possible factors affecting this glycerol formation. In response to the chemical elicitor A. niger pectinase, both the cytoplasmic and vacuolar pH of C. roseus hairy roots decreased immediately, and phosphate uptake by the hairy roots was hampered. These initial responses were followed by a shift in carbon fluxes as indicated by the transient increases in alanine and succinate, and decreases in glutamine and pyruvate. Glucan synthesis, malate and ethanol accumulation were significantly enhanced by the elicitor treatment. The Ca$sp{+2}$ ion is an important factor in the glucan synthesis. Alkaloid analysis showed enhanced ajmalicine and catharanthine production by the A. niger pectinase-treated C. roseus hairy root cultures.

[发布机构] Rice University