Correlation Between The Diversity Of Endophytic Fungi And Taxoids Accumulation In Taxus Cuspidata And The Application Of The High-yield Strain

As one of the most ancient species derived from quaternary glaciation.Taxus chinensis are recognized as endangered plants around the world with universally acknowledged antitumor activity,which are regarded as 'living fossil' in the plant kingdom Taxus chinensis contains various bioactive components,including taxoids,flavonoids,lignans,steroids,phenolic acids,sesquiterpenes and glycoside.Among them,paclitaxel is most widely used broad-spectrum anticancer drug in clinical all over the world.However,the proportion of natural paclitaxel in Taxus chinensis is only 0.02-0.069%,which is difficult to meet the market demand.Since the firstly identified and isolated from Taxus brevifolia in 1993,taxol-producing endophytic fungi has become critical research object as a replacement of natural Taxus chinensis,for the obtained of paclitaxel in vitro.According to the reports,endophytic fungi and their secondary metabolites play significant roles in the development and bioactive compounds accumulation of their host.In the present study,Taxus cuspidal a Sieb.et Zucc.was used as research object,which components were analyzed quickly and comprehensively by UPLC-Q Exactive Focus Orbitrap/MS technology.The iconic metabolites from four parts of Taxus cuspidata were analyzed and quantified combined with UPLC-MS/MS,and the correlations between endophytic fungal and metabolites of the host were systematically explored.In this study,several new ideas for the development and utilization of new natural product resources and medicinal plants.The results are as follows:1.Taxonomic identification and species diversity analysis of endophytic fungi from different parts of Taxus cuspidataA total of 262 endophytic fungi were isolated from 4 organ parts(roots,branches,needles and fruits)of Taxus cuspidata,including 98 in roots,86 in branches,69 in needles,and 9 in fruits.Fungal were identified based on the morphology and ITS sequences.The species richness and diversity of endophytic fungi were significantly affected by tissues.The diversity and abundance of endophytic fungi in roots and branches were 1.91-2.36 and 17.19-21.37 folds higher that in needles and fruits,respectively.Trichoderma was unique advantages of different tissue parts.The most dominant orders i.e.Eurotiales,Pleosporales and Hypocreales and the most dominant genera Penicillium,Aspergillus,Allernaria,Fusarium,Xylaria,Botrytis.Phoma and Trichoderma showed obvious tissue-specificity.Most dominant species show obviously tissue specificity,and many fungal with low isolation frequency also show tissue specificity.2.Study on the global metabolic components of Taxus cuspidata by plant metabonomics technologyA comprehensive analysis of the metabolic components of Taxus cuspidata,combined with mass spectrometry cleavage patterns,12466 ion characteristics,corresponding to 5498 annotated potential metabolites.There were identified 239 compounds including flavonoids,terpenes,phenylpropanoids,alkaloids,organic acids,sugars,amino acids,etc.These metabolites participate in 80 metabolic pathways,mainly including amino sugar and nucleotide sugar biosynthesis pathway,terpenoid backbone biosynthesis pathway,fatty acid biosynthesis pathway,flavonoid biosynthesis pathway,phenylpropane biosynthesis pathway,and steroids biosynthesis pathway,citric acid cycle(TCA cycle),anthocyanin biosynthesis,phenylalanine metabolism pathway,flavonoid and flavonol biosynthesis pathway,isoquinoline alkaloid biosynthesis pathway,etc.The total amount of taxoids in Taxus cuspidata were the highest,and taxoids were distributed throughout the parts of the yew plant,and the content and species distribution of different parts were quite different.Differential analysis of the precursor,intermediate products and taxoids metabolites of the paclitaxel biosynthesis pathway,combined with UPLC-MS/MS to compare seven target taxoids for analysis and detection.It could be speculated that the synthesis,transportation and accumulation of taxoids in plant tissues and organs.3.Correlation analysis of different tissues typical metabolites and endophytic fungi in Taxus cuspidataThe relationship between chemical composition and endophytic fungi of T.cuspidata were investigated by correlation analysis and linear regression analysis models.The results showed that the content of active ingredients in different tissues of T.cuspidata had significant correlation with the species richness of endophytic fungi,and it was found that there was a positive correlation between the dominant genera and the content,especially Epicoccum and paclitaxel.The content of 21 differential compounds were associated with some tissue-specific endophytic fungi,and there were positive and negative correlations between them,indicating that endophytic fungi played an important role to accumulate and produce compounds of Taxus cuspidata.The endophytic fungi of T.cuspidata in different tissues have the functions of promoting plant antibacterial,antiviral,and antioxidant functions,but some fungi cause diseases and accelerate tissue decay when plants fall into decay period.4.Optimization of fermentation process for endophytic fungus R8-3-4 of Taxus cuspidataPCR amplification of key biosynthetic enzyme genes and UPLC-MS/MS method were used to screen the target strains which can produce the same bioactive components as the host from the endophytic fungi in the root bark of T.cuspidata.R8-3-4 strain was used as the research object to investigate the effects of different factors on the yield of 10-DAB.The optimal fermentation conditions for the production of 10-deacetylBaccatine ? as follow:PDB medium,carbon source glucose,initial pH 6.0,temperature 30?,rotation speed 160 r/min,culture for 14 days,CuSO4 0.10 mg/L,salicylic acid 10 mg/L,sodium acetate 8 g/L,the 10-DAB yield of strain R8-3-4 after fermentation optimized culture was 983.41?g/L,which was 2.96 times the yield before optimization.Magnetic immobilization fermentation was used for semi-continuous production of 10-DAB.After five cycles,the final total concentration of 10-DAB in the 5 L bioreactor reached 4873.08 ?g,realizing a huge economically sustainable expected possibility on a commercial scale.Green environmental protection and sustainable production strategy is expected to solve the contradiction between supply and demand of active products of paclitaxel,which has forward-looking strategic value for the production and research of natural products.