Abstract
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.
Highlights
With NASA support this research aimed at evaluating early opportunities in Microgravity Sciences to commercialize space and to develop the biotechnology facility for the International Space Station [1]
The main task was to evaluate the production of taxol with cell suspensions in bioreactors designed for the Space Shuttle
This work led to the early demonstration of L-arginine-dependent nitric oxide (NO) bursts in mechanically and gravitationally stressed plant cells, to NO-induced programmed cell death, and to a model describing how these factors contribute to increased taxol and taxane recovery from conifers
Summary
With NASA support this research aimed at evaluating early opportunities in Microgravity Sciences to commercialize space and to develop the biotechnology facility for the International Space Station [1]. In our work with Taxus cell suspensions, the substituted guanidines offered protection against mechanically induced stress and celldeath or apoptosis. The use of NO donors, NOS substrates, products, inhibitors (substituted guanidines), and NO traps provided new opportunities to control the citrulline-NO cycle (See Figure 1), apoptosis, and taxol production in unit gravity, simulated microgravity, and in hypergravity. Reactions 7 and 8 comprise decarboxylation, oxidation, methylation, transamidination, phosphorylation, keeping the guanidino group intact or modifying it by methylation, phosphorylation, etc They remove L-arginine as a substrate from the urea cycle, and from reactions 5 and 6. The stress-induced NOS activity links respiration to NO, ROS and RNS production, their signaling pathways and damaging reactions, e.g., the nitration of phenols and tyrosines residues in cell regulatory proteins, and to apoptosis. For samples larger than 10 g fresh biomass, these compounds were determined by HPLC using authentic standards and taxil columns (MetaChem Technologies Inc.) [12]
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