Research On The Effect On Key Enzymes And Biosynthetic Yield Of Epothilone

The epothilones are a novel class of antineoplastic agents,their molecular structures having a variety of slight differences but similar efficacy were identified as 16-member macrolides.Their names come from structural subunits Epoxid,Thiazole ring and Ketone.The epothilones were originally identified as secondary metabolites produced by the myxobacterium Sorangium cellulosum,epothilones induce tubulin polymerization and enhance microtubule stability,the effect is similar to paclitaxel some derivative of these compounds were developed new product.Epothilones have high water solubility and pharmaceutically active,wide selectivity,low toxicity,molecular structure is simpler than paclitaxel,broad-spectrum anti-tumor,treating multi-drug resistant cells with high anti-cancer activity and so on,they have high application value and development potential in medicine industries.Pyruvate kinase?PK?,S-adenosylmethionine synthetase?SAMs?and ?-ketoglutarate dehydrogenase complex?OGDHC?are key enzymes in metabolic pathways of epothilones biosynthesis.Vitamin K₃?VK₃?and Fructose-1,6-diphosphate?FDP?have a effect of inhibition and activation on Pyruvate kinase respectively.Sodium hypochlorite?NaClO?and Hydrogen peroxide?H₂O₂?are inhibitor of ?-ketoglutarate dehydrogenase complex.P-toluene sulfonate?p-TSA-Na?and Indole acetic acid?IAA?have a effect of activation and inhibition on S-adenosylmethionine synthetase respectively.Change tendency of these fermentation parameters are measured to research of effector influence on key enzymes and biosynthetic yield of epothilone.These parameters are the specific activity of key enzymes,yield of epothilone biosynthesis,dry cell weight within the fermentation period,pH and reducing sugar content of the medium,biosynthetic yield of epothilone per unit mass of DCW.Relative expression levels of target gene measured by Q-PCR.It was found that four kinds of inhibitors have different influence on fermentation parameters and two activators did.Inhibitory effect of IAA and VK₃ was obvious and temperate but NaClO and H₂O₂ is fierce and abysmal.Synthetically speaking,activation effect of p-TSA-Na is superior to FDP.Adding sodium p-toluenesulfonate to the medium had a negligible effect on cell growth.Biosynthetic yield of epothilones and concentrations of sodium p-toluenesulfonate were positively correlated within the range of 1³2 mmol/L,the range from 4 mmol/L to 16 mmol/L was most obvious.The promotion of biosynthetic yield of epothilones by adding applicable concentrations of sodium p-toluenesulfonate was vertified.Subsequent experiments proveded that inoculation was the best time to add sodium p-toluenesulfonate to the medium.when the initial concentration of the culture medium at 24 mmol/L,biosynthetic yield of epothilones had a maximum,after verification of yield stability,yield of EpothiloneA reached 10.31mg/L and Epothilone B reached 6.63mg/L,experimental group increased by 49.64% and 43.82% compared to the yield of the control group,respectively.At the same time,activity of S-adenosylmethionine synthetase raised the range from 22% to 75%.Adding 24mmol/L sodium p-toluenesulfonate to the medium immediately after inoculation can account for the biosynthetic yield of epothilones having a maximum,activity of S-adenosylmethionine synthetase is also improved appreciably.This research provided valuable information to practical production.Biosynthesis mechanisms of epothilones by adding sodium p-toluenesulfonate need more depth and comprehensive research in order to improve the biosynthetic yield of epothilones.