Isolation,Identification And In Vitro Anticancer Bioactivity Of Taxol-producing Endophytic Fungus Alternaria Alternata F3 From Taxus Cuspidata

Taxus cuspidata is a member of the genus Taxus.It is mainly distributed in Northeast China,Japan,South Korea,North Korea,and the Russian Far East.It has been widely studied because it can produce the first-line anticancer drug paclitaxel.Taxus chinensis has extremely low content of paclitaxel.Currently,searching for new sources of paclitaxel has become a research hotspot.Among many methods,microbial fermentation is an important way to obtain taxol.It can solve the current dependence on taxus plants.Endophytic fungi are microorganisms that live in the internal tissues of a healthy host,can coexist with plants for a long time,usually do not cause any damage to the plant,and can produce metabolites with the same or similar structure as the host.Therefore,endophytic fungi can be used as an important source of secondary metabolites.In this study,endophytic fungi were isolated from healthy Taxus cuspidata plants,and the production of paclitaxel was screened by thin layer chromatography(TLC),high performance liquid chromatography(HPLC)and ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The fermentation conditions were optimized to maximize the yield of the fungal taxol.Finally,the biological activity of the endophytic fungus extract was studied.The main research results are as follows:1.A total of 84 endophytic fungi were isolated from different tissues of Taxus cuspidata.According to the different forms of endophytic fungi,the isolated fungi are classified into the following categories:Fusarium sp.,Epicoccum sp.,Alternaria sp.,Phoma sp.,Geotrichum sp.,Didymella sp.,Coniothyrium sp.,Paraconiothyrium sp.,Peyronellaea sp.,Daldinia sp..2.TLC,HPLC and UPLC-MS/MS methods were used of analyze the methylene chloride extract of fungi to identify whether the crude extract contains paclitaxel.For TLC detection,the developing solvent was chloroform/methanol(5:1,v/v),and UV254 nm was as the developed color.It was found that a crude of fungal F3 extract had the same color spots at the same position with the paclitaxel standard.The Rf value was 0.75.The fungal extract was further analyzed by HPLC.HPLC detection conditions are as fellows:Column was HiQ sil C18W(4.6 mm×250 mm,5 μm),column temperature was at 30℃,mobile phase was acetonitrile and water,detection wavelength was at 227nm,flow rate was lml/min.The same chromatographic peak appears between the fungal F3 extract and the paclitaxel standard at 26.7min.Using UPLC-MS/MS to identify the taxol in the F3 fungal extract,it can produce the same molecular ion peak of m/z of 876 as paclitaxel standard in 1.7 min.The molecular ion peak is further analyzed,the fungal F3 extract and the standard paclitaxel can also produce an ion peak of m/z 308,which proves that the endophytic fungus F3 can produce paclitaxel characteristically.Through morphological observation and molecular biology identification,the F3 strain was determined to Alternaria alternata.3.The single-factor experiment(different culture media,inoculation amounts,initial pH,temperature and culture time)were optimized to enhanceing the yield of fungal taxol.Through the combination of PB experiment and CCD experiment with RSM,the optimal concentration of precursor and inducer was screened.Finally,the optimal culture condition of the fungus Alternaria alternata was as follows:the medium was YPD,the inoculum amount was 2%,the initial pH of the medium was 6.0,and the culture was cultured.At a temperature of 28℃,the 0.1 g/L sodium acetate,0.25 g/L salicylic acid and 0.00125 g/L silver nitrate was added to the medium;under the optimal culture conditions,the yield of paclitaxel is 195.4±0.23 μg/L,which is 2.17 times of the original culture conditions.4.Using the MTT method,the effect of the taxol-producing Alternaria alternata F3 was explored on the cell viability of lung cancer A549 and cervical cancer HeLa cells.The experimental results showed that the fungal paclitaxel inhibited the proliferation of A549 and HeLa cells in a time and dose dependent manner.The inhibitory effect of fungal extract on A549 had IC50 of 26.36 μg/mL,23.89 μg/mL,15.95 μg/mL at 24h,48h,and 72h,respectively.When Hela cells were treated with the same concentration of fungal extract,the IC50 was 14.24μg/mL,11.15 μg/mL,11.70μg/mL at 24h,48h,and 72h,respectively.In summary,a total of 84 endophytic fungi were isolated from Taxus cuspidata.A specific taxol-producing endophytic fungus Alternaria alternata F3 was selected.The production of paclitaxel is increased through fermentation optimization.Through the anti-cancer activity of the fungal paclitaxel extract,it was confirmed that the fungal extract had a significant inhibitory effect on the survival rate of A549 and HeLa cells.It provides a strain foundation for the future using molecular biology methods to improve and cultivate new varieties of taxol-producing endophytic fung.The research results of this thesis have enriched the new drug source of taxol,which is worthy of development and utilization.