Searching The Genetic Evidence For The Capability Of Taxol Synthesis In Several Materials

Taxol(paclitaxel), a complex diterpenoid first isolated from the bark of pacific yew tree(Taxus brevifolia), is widely used in chemotherapy treatment of lung, ovarian and breast cancer. The supply of taxol is currently constrained and supplied by a number of routes including harvesting from relatively slow-growing Taxus trees. Thus, alternative sources for taxol have been actively explored for the past 20 years, including a search for taxol-producing microorganisms.Diversity of endophytic fungi of Taxus spp. have been explored by isolating culturable microorganisms on culture medium, but non-culturable endophytic isolates cannot be found using this conventional methods. Next generation sequencing technologies have enabled metagenomic and metagenetic analysis of soil microorganism species and gene composition of microbiota. However, there are currently no studies characterizing species and gene composition of root associated microbiome of the roots from Taxus. In this study, we used bar-coded high-throughput sequencing with primers targeting the 16 S and 18 S rDNA genes to survey root associated bacterial and fungal diversity of Taxus root, in conjunction with a metagenome approach to survey microbial species and gene composition in its root associated microbiome. We also studied genes putatively associated with taxol biosynthesis in the Taxus root associated microbiome to estimate the prevalence of taxol biosynthetic genes in the root associated microbiome. Through these studies we hope we can find some taxol producing microbes species.Besides taxane-producing endophytes, taxanes including taxol had been found from Yunnanopilia longistaminata, Cephalotaxus sinensis, Corylus heterophylla Fisch, Pinus massoniana Lamb and Podocarpus macrophyllus. But there are still not enough evidences to prove the taxanes producing ability in these species. If the taxadiene synthease gene was found in those species, that will be the best evidence to support their taxanes producing ability. So we studied the genome or transcriptome data of Penicillium aurantiogriseum NRRL 62431, Y. longistaminata and C. Sinensis, and identified several taxadiene synthease like genes. From our experiments, we found that genes predicted as TSL have no taxadiene synthase activity in vitro. The main studies are as follows:1. In the second chapter, we surveyed the microbial diversity and gene composition of the root-associated microbiome from T. chinensis(Pilger) Rehd. From the 16 S bacterial library, 640 OTUs were identified. Our study is the first report of Proteobacteria being dominant in the root of T. chinensis. Unfortunately, we didn’t find any reported taxol-producing bacterial species. Through analysis of the sequences obtained from the 18S-derived library, 187 OTUs were identified. We found Hyphodontia, Hemimycena, Phialocephala are the three dominant genera, and to our knowledge, this is the first time these three genera were reported in any Taxus sp. Our high-throughput amplicon sequencing revealed 187 fungal OTUs which is higher than any previously reported fungal number identified with the culture-dependent method, suggesting that T. chinensis roots harbor novel and diverse fungi. Metagenomic analysis that consisted of sequencing a DNA library derived from the T. chinensis root DNA showed that Alphaproteobacteria, Gammaproteobacteria and Bacilli were the most dominant bacterial phyla, and Saccharomycetes, Glomeromycetes and Sordariomycetes were the dominant fungi. Five bacterial genera(Erwinia, Curtobacterium, Pantoea, Bacillus and Sphingomonas) were reported to have species with taxol-production capacity.2. In the third chapter, we analyzed the genome data of a taxol producing fungi P. aurantiogriseum NRRL 62431, and found a GGPPS gene and a TS like gene(TSL). In section one, we cloned the ORF fragment of the PaTSL gene, and then we studied the activity of the protein encoded by PaTSL. But no taxadiene was found in the product of the reaction, so PaTSL gene maybe not taxadiene synthease gene, or the enzyme encoded by it had no taxadiene synthease activity in vitro. In section two, we cloned the ORF fragment of PaGGPPS. The bioinformatics methods were employed to analyze and predict the composition of nucleic acid and amino acid sequences of this gene. After the bioinformatics analysis, we constructed a pET30 a prokaryotic expression vector of PaGGPPS. The protein of PaGGPPS was successfully expressed in the soluble state in Escherichia coli. All the experiments we had done can provide some information for the further research.3. In the fourth chapter, we carried out a series of studies on Y. Longistaminata, in which taxanes were detected. In section one, transcriptome sequencing was performed on Y. Longistaminata. Nearly 4.8Gb sequence data and 51,744 unigenes were obtained, and a total of 6,513 genes were mapped into 335 KEGG pathways. Interestingly, 41 unigenes were mapped into the terpene biosynthesis pathway. Using local blast, we found 2 predicted TS gene fragments. In section two, we cloned the full length of YlTSL1 using RACE-PCR, and then we studied the activity of the protein encoded by YlTSL1. No taxadiene was found in the products of the reaction, so we tested YlTSL1 in another expession system, and identified it is α-farnesene synthase gene. In section three, we cloned the full length of YlTSL2 using RACE-PCR, and then studied the activity of the protein encoded by YlTSL2. YlTSL2 were identified as kaurene synthase gene.4. In the fifth chapter, we studied transcriptome data of C. Sinensis, in which Luo et al. had found taxanes. We cloned the full length of CsTSL using RACE-PCR, and then we studied the activity of the protein encoded by CsTSL. Verticiol and 17-methyl-5a-androst-2-ene were found in the products of the reaction, but no taxadiene was found. 17-Methyl-5a-androst-2-ene was a hormone analogs, and was the major product of the reaction. Erdtman postulated that verticiol was the intermediate products in the taxadiene synthase catalyzed reaction. The mechanism of the taxadiene synthase-catalyzed cyclization of GGPP to taxadiene is proposed to proceed through a verticillen-12-yl carbocation intermediate that undergoes an 11- 7 proton transfer leading to formation of the C ring.