Nitric oxide, cell death and increased taxol recovery.

Genes for plant nitric oxide synthase (NOS) now help explain how NO is produced from nitrate, nitrite, and/or L-arginine in the culture medium and in cells. Evidence is presented that under aseptic conditions; plant NOS activity, NO bursts, and cell death (apoptosis) are important factors in the recovery of taxol (paclitaxel) from cell suspensions of several Taxus sp. Cell-suspension responses to mechanical stresses, simulated microgravity, and hypergravity were dominated by NO bursts and cell death (apoptosis) and by the overproduction and release of free and bound taxol into the culture medium. The synthesis of the taxane ring in the chloroplasts and gravisensing amyloplasts, and the subsequent assembly of taxol and related taxane diterpenoids were visualized by immunocytochemical, laser confocal and scanning electron microscopy. Drug-producing cells and taxane-bearing materials in the culture medium were recovered using immunoparamagnetic beads. Bound taxol and taxanes were recovered from xylanase hydrolyates. Binding was associated with the expression of two proteins for ‘touch’ genes expressed under mechanical forces. Taxol recovery was increased by 64% by the use of a NO donor. NOS inhibition with guandino compounds, and the use of a NO trap, reduced taxol recovery and reduced taxane diterpenoid biosynthesis. This offered countermeasures to NO-mediated stress, and reduced apoptosis. In hypergravity, the taxol and taxanes released from cells by syneresis were recovered on hydrophobic PVDF (polyvinylidene fluoride) filters. Cyclodextrins added to the culture medium enhanced biomass yield and altered the solubility of taxanes to improve the recovery of taxol and taxanes.