Study On Mechanism For Mechanotransduction And Metabolic Adaptations Of Suspension Taxus Cells Under Shear Stress

Understanding the mechanism for the effect of shear stress on plant cell has great value for plant cell culture reactor design and scale and operation optimization, the research strategy of this work is to culture suspension Taxus cells in the uniform laminar shear reactor. The role of phospholipid signaling in the mechanotransduction processes was revealed by analyzing the changes of phospholipid species. The role of cell membrane in the mechanotransduction processes was investigated by analyzing the changes of membrane lipid composition. The defensive mechanism that enabled plant cells to adapt to shear stress was studied by analyzing the changes of primary metabolites. The potential strategies to increase the yields of paclitaxel and other desired taxoids were explored by analyzing the changes of secondary metabolites.The systematic analysis of changes in phospholipid species in shear treated Taxus cells was based on LC/ESI/MS. The results of PCA and PLS-VIP analysis revealed that phosphatidic acid (PA) and lysophosphatidylcholine (LysoPC) were two important lipid groups which were responsible for the discrimination between shear stress induced and control cells. And the contents of PA and LysoPC increased significantly after shear stress treatment. The results of inhibitor experiments revealed that shear stress enhanced the activation of phospholipase D (PLD) and phospholipase C (PLC) compared with control cells and consequently increased PA content in shear stress induced Taxus cells, which revealed the activation of phospholipid signaling in mechanotransduction of Taxus cells in response to shear stress. The mechanism for shear signaling transduction by interconversion of phospholipids was discussed.The systematic analysis of changes in membrane lipid composition (phospholipids, sterols, and fatty acid) in shear treated Taxus cells was based on LC/ESI/MS and GC-TOF-MS. The main changes of membrane composition induced by shear stress were the contents of structural phospholipids decreased; cells showed increased stigmasterol at the expense of sitosterol and campesterol; the stigmasterol/phospholipid ratio increased; the contents of mono-unsaturated-fatty-acid and very long-chain saturated fatty acids increased. The results indicated the changes of cell membrane to adapt to shear stress and further revealed the molecular basis of shear signal transduction processes. The systematic analysis of changes in primary metabolites in shear treated Taxus cells was based on GC-TOF-MS. Potential biomarkers were found by PCA as well as PLS-VIP. Trehalose, sorbitol, ascorbate, sucrose, and gluconic acid were mainly responsible for the discrimination between shear stress induced cells and control cells. Further analysis by mapping measured metabolite concentrations onto the metabolic network revealed that shear stress imposed restrictions on primary metabolic pathways by inhibiting tricarboxylic acid cycle, glycolysis, and N metabolism. To adapt to the shear condition, cells responded by starting defensive programs. These defensive programs included coinduction of glycolysis and sucrose metabolism, accumulation of compatible solutes, and antioxidative strategy. The possible shear defensive mechanism from the view of metabolites was proposed.The systematic analysis of changes in secondary metabolites in shear treated Taxus cells was based on UPLC/Q-TOF MS and LC/ESI/MS. It was found that the contents of taxol and detected intermediates involved in paclitaxel biosynthesis were all reduced, and the contents of most uninvolved taxoids were also decreased except several classes. Analysis of mapping measured taxoids concentrations onto paclitaxel biosynthesis pathway illustrating proposed intermediates and“off-pathway”metabolites revealed that shear stress might disrupt the appropriate cyclization process of GGPP, aggravate the inappropriate order of hydroxylations and acylations, and not be good for functional group oxetane formation. The possible mechanism for shear stress limiting production of taxol by Taxus cells was revealed.