Genetic Transformation Of Taxol-Producing Endophytic Fungi And Molecular Cloning Of Genes Involved In Taxol Biosynthesis Pathway

Taxol is one kind of highly effective, low cytotoxic and broad-spectrum natural anticancer drugs. Nowadays, Taxol is mainly isolated from the bark of yew tree or semi-synthesized via precursor baccatin III, which is also isolated from the yew tree. However, due to the relative scarcity of yew trees, slow growing rate and extremely low content of Taxol in the taxus, the supply of Taxol is very limited. Therefore, it is urgent to find other alternative approaches to increase the supply of this promising anticancer drug. Producing taxol through fermentation of taxol-producing fungi may be one of the promising ways to solve the Taxol supply problem.In order to solve the Taxol supply problem, this study focuses on two aspects. The first is the isolation of taxol-producing endophytic fungi from the bark of wild yew tree, the development of an efficient transformation system for Taxol-producing endophytic fungus, and the successful construction of fungal expression vectors involved in taxol biosynthetic pathway genes in order to make a basis for improving Taxol production of endophytic fungi by genetic engineering of the taxol-producing endophytic fungi in the near future. The second is the molecular cloning, characterization and functional analysis of related genes involved in Taxol biosynthesis pathway from a new Taxol-producing plant species, Corylus avellana L. Gasaway. This will provide some candidate genes for hazel genetic engineering to improve Taxol yield in the future. The main results from the study are as follows:1) By surface sterilization, 120 different endophytic fungi strains were isolated from wild Taxus chinensis var. mairei inner bark. No. EFY-110 strain was detected to produce both Taxol and baccatin III by HPLC and LC-MS analyses. The biological activity assay showed that the culture extract, like the standard sample of Taxol, could also effectively kill or inhibit liver cancer cells BEL7402. The fungus strain was different from that of the endophytic Taxol-producing fungi previously reported, and was identified as Didymostilbe sp. according to its morphology and growth rate of the colony, as well as the characteristics of the mycelia and conidia, and was named DF110.2) A full-length cDNA encoding 3-hydroxy-3-methylglutaryl-CoA reductase gene (designated as BT2HMGR) and its genomic DNA sequence were cloned and characterized from the endophytic Taxol-producing fungus BT2 previous reported in our group by using rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of BT2HMGR was 3926 bp containing a 3522 bp ORF encoding 1173 amino acids. The comparison between cDNA and genomic DNA sequences revealed that BT2HMGR was composed of four exons and three introns. BLASTP analysis revealed that the deduced BT2HMGR had extensive similarity with other fungi HMGRs. Southern blot analysis showed that BT2HMGR was a single copy gene. RT-PCR analysis indicated that BT2HMGR could be induced by methyl jasmonate (MeJA). 3) An efficient transformation system for BT2 has been successfully developed by REMI technique. After hygromycin selection, 42 hygromycin-resistant colonies were regenerated. PCR and Southern blot analyses further confirmed that the foreign gene (hph) in the transforming plasmid was successfully integrated into the chromosomal DNA of BT2. The transformation efficiency was about 5-6 transformants/μg pV2 DNA. This study provides a basis for improving Taxol production by engineering Taxol-producing fungi with Taxol biosynthesis pathway genes in the future.4) Four monovalent fungi expression vectors (pV2+TS, pV2+DBAT, pV2+BAPT and pV2+DBTNBT) which contained the key enzyme genes (TS, DBAT, BAPT and DBTNBT) involved in Taxol biosynthesis pathway were firstly constructed. This study provides candidate expression vectors for improving Taxol-producing fungi by genetic engineering in the near future.5) The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step of terpenoids biosynthesis in MVA pathway. The full-length cDNA of HMGR gene was isolated from hazel (Corylus avellana L. Gasaway), a Taxol-producing plant species (designated as CgHMGR) by RACE for the first time. Sequence analysis showed that CgHMGR had high homology with other plant HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small multi-gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis ofβ-carotene, indicating that CgHMGR encoded a functional protein.6) A full-length cDNA encoding isopentenyl diphosphate isomerase was first cloned from hazel (Corylus avellana L. Gasaway) (designated as CgIPI) by RACE technique. Sequence analysis revealed that the CgIPI had high homology with other plant IPP isomerases. Phylogenetic tree analysis revealed that CgIPI shared the same ancestor with IPIs from other plant species. Southern blot analysis indicated that CgIPI belonged to a small multi-gene family. Tissue expression pattern analysis showed that CgIPI expressed higher in roots than in stems and leaves. The functional color assay indicated that CgIPI could also accelerate the accumulation ofβ-carotene in Escherichia coli transformants, demonstrating that the CgIPI encoded a functional protein.7) A full-length cDNA encoding geranylgeranyl diphosphate synthase, which catalyzes the formation of GGPP by condensation of IPP with a farnesyl pyrophosphate (FPP), was isolated from (Corylus avellana L. Gasaway) (designated as CgGGPPS) by RACE. The deduced CgGGPPS was found to have extensive homology with other plant GGPPS proteins via multiple alignments. Sequence analysis indicated that CgGGPPS contained seven highly conserved prenyltransferases domains (I-VII). Phylogenetic analysis showed that CgGGPPS had the closest relationship with GGPPS from H. brasiliensis. Southern blot analysis indicated that CgGGPPS belonged to a small multi-gene family. Tissue expression analysis showed that CgGGPPS expressed in roots and stems at a similar level, but the expression was higher in leaves. Expression of CgGGPPS could be induced by methyl jasmonate (MeJA).In summary, the isolation, taxonomic identification, culture, extraction and product detection of a new Taxol- and baccatin III-producing endophytic fungus DF110 (Didymostilbe sp.) were first reported. BT2HMGR gene involved in Taxol biosynthesis pathway was first isolated from the endophytic Taxol-producing fungus strain BT2. Furthermore, an efficient transformation system for BT2 was established by REMI technique and four fungi expression vectors containing taxol biosynthetic pathway genes were constructed. Meanwhile, the cloning of three genes (CgHMGR, CgIPI and CgGGPPS) from hazel was also reported in the present study. The genomic organization, expression profiles and functional identification of the cloned genes were also studied. This study provides a basis for genetically engineering Taxol-producing endophytic fungi and hazel for improved Taxol yield in the future, and will be helpful to understand more about Taxol biosynthesis and regulation mechanism in Taxol-producing endophytic fungi and hazel at molecular level.