Isolation of endophytic fungi from Taxus spp. plant material
Endophytic fungi were isolated as previously described by Guo et al. (2006). Bark segments (0.5 × 0.5 cm) were removed with a sterile scalpel and surface sterilized for 5 min in 70 % ethanol. The inner bark was separated from the outer layer and placed on PDA agar (Carl Roth GmbH, Karlsruhe, Germany) supplemented with 25 mg/L streptomycin. The plates were incubated at room temperature until fungal growth was visible. The mycelium was then transferred to fresh plates using the hyphal tip method.
Cultivation of endophytic fungi
The isolated endophytic fungi were cultivated on solid media, PDA (Carl Roth GmbH) supplemented with streptomycin or on YM-6.3 agar (0.4 % (w/v) glucose, 0.4 % (w/v) yeast extract, 2 % (w/v) malt extract, pH 6.3, 1.5 % (w/v) agar-agar). The fungi were transferred to fresh plates at weekly intervals by cutting out a piece of overgrown agar. In liquid culture, the fungi were grown in 0.6–10 L YM-6.3 medium (120 rpm in the dark) for 3 weeks or until no more glucose could be detected. The fungi were also cultivated in S7 medium as described for taxane-producing endophytes (Stierle et al. 1993).
For taxane analysis, the fungal culture media were extracted twice with an equal volume of chloroform. The organic phase was then dried over magnesium sulfate, evaporated to dryness and the residue was redissolved in 3–5 mL methanol. Plant material (30 g Taxus needles or tobacco leaf tissue) was lyophilized and extracted with 1:1 dichloromethane/methanol in a Soxhlet extractor. The organic solution was evaporated to dryness and redissolved in dichloromethane. After two rounds of extraction with water, the organic layer was dried over magnesium sulfate, evaporated to dryness and the residue was redissolved in methanol (Witherup et al. 1990).
Anti-taxane immunoassay (competitive inhibition enzyme immunoassay, CIEIA)
The anti-taxane immunoassay was carried out according to the manufacturer’s instructions (Cardax Pharmaceuticals, Hawaii). A standard curve for taxane quantitation was made using Taxol concentrations of 111, 37, 12.33, 4.11, 1.37, 0.46 and 1.15 ng/mL (Table S1). The samples were analyzed using three dilutions. Values in the linear range of the standard curve were used to calculate the concentration.
LC/MS/MS was carried out in multiple reaction monitoring scan mode using a QTrap3200 system (Applied Biosystems, Darmstadt, Germany). The three most intensive mass transitions for three standard substances (Taxol, baccatin III and 10-deacetyl-baccatin III; Sigma-Aldrich, Idena) were used for detection (Table S2). Analysis in ESI negative ionization mode was carried out using the following settings: curtain gas 25 psi, CAD gas medium, ionspray voltage −4,500 V, temperature 450 °C, gas1 50 psi, gas2 65 psi. HPLC separation was carried out using a Curosil PFP column (150 × 3 mm, 3 μm; Phenomenex, Aschaffenburg, Germany) under the following conditions: column oven, 25 °C; LC flow rate, 300 μL/min; solvent A, 98 % water and 2 % acetonitrile with 10 mM ammonium acetate; solvent B, 2 % water and 98 % acetonitrile with 10 mM ammonium acetate; gradient, 0 min 70 % A, 0.5 min 70 % A, 15 min 0 % A, 20 min 0 % A, 21 min 70 % A, 23 min 70 % A.
DNA isolation, construction of genomic phage libraries and hybridization
Fungal and plant genomic DNA was isolated using a modified CTAB method. Plant and fungal samples (1 g) were homogenized with a mortar under liquid nitrogen, supplemented with 10 volumes of CTAB buffer (100 mM Tris pH8, 20 mM EDTA, 1.4 M NaCl, 2 % β-mercaptoethanol, 2 % CTAB) and incubated for 1 h at 65 °C. The cell debris was removed by centrifugation (15 min, 2,000 × g) and the supernatant was extracted twice with an equal volume of 24:1 chloroform:isoamylalcohol. The DNA was then precipitated with isopropanol. Genomic phage libraries were constructed from EF0001, EF0021 and Taxomyces andreanae DNA, and plaque lifting was carried out according to the manufacturer’s guidelines (Lambda Dash® II / Gigapack® III XL, Stratagene). Heat-fixed membranes (Nylon N+, GE Healthcare) were supplemented with 20 mL Roti-Hybri-Quick (Carl Roth GmbH) and 100 μg/mL salmon sperm DNA (Sigma) in hybridization rolls. Pre-hybridization was carried out for 3 h at 55 °C. Probes against taxadiene synthase (TDS) and taxane-13α-hydroxylase (T13H) were prepared by PCR using primers corresponding to specific target genes, i.e. TDS1 (forward 5′-GCA GCG CTG AAG ATG AAT GC-3′, reverse 5′-CGA TTC GAT ACC CCA CGA TCC-3′, bp 22–546), TDS2 (forward 5′-GCC CTC GGC CTC CGA ACC C-3′, reverse 5′-GCC ATG CCG GAT TCT TTC CAC C-3′, bp 1,211–1,710), TDS3 (forward 5′-GGT GGA AGG AAT CCG GCA TGG CAG-3′, reverse 5′-GTC GCC AGC TCA AGG ATA CAA GCT C-3′, bp 1,693–2,263) andT13H (forward 5′-ATG GAT GCC CTT AAG CAA TTG GAA GTT TCC CC-3′, reverse 5′-GCT CCT GCA GGT GCT CC-3′, bp 1–604). The reactions were heated to 94 °C for 2 min followed by 25 cycles of 94 °C for 30 s, 55–60 °C for 30 s, 72 °C for 45 s and finally 72 °C for 5 min. Incorporation of α32P-dATP (Hartmann Analytic, Braunschweig, Germany) was done using the Hexalabel™ DNA Labeling Kit (Fermentas, St. Leon-Rot, Germany). The TDS and T13H probes were purified on G-50 gel filtration columns (GE Healthcare, Karlsruhe, Germany). Probes against taxane-5α-hydroxylase (T5H) were prepared by labeling oligonucleotides with γ-32P-dATP using polynucleotide kinase (oligo1 5′-GGC ATC CCA CAG TAG TAC TCT GCG GCC CTG CGG GAA ACC GGC TTA TTC TGT CCA ACG AGG AGA AGC TGG TGC AGA TGT CG-3′, and oligo2 5′-CCA CCA CTT CGC CAA TGG CTT TGA TTT TCA AGC TCT TGT CTT CCA ATC CAG AAT GCT ATC AAA AAG TAG TTC AAG AGC-3′). Probes were added to the pre-hybridization mix and hybridized against the membranes overnight at 55 °C. The membranes were washed three times for 30 min with 1:2, 1:5 and 1:10 dilutions of hybridization buffer, and then visualized by autoradiography on pre-flashed X-ray films (Hyperfilm MP, GE Healthcare) at −80 °C for 2 days.
Amplification of internal transcribed spacer (ITS) sequences
ITS regions from the isolated Taxus endophytes were amplified by PCR using the universal primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (Sim et al. 2010) in 2× PCR-MasterMix Solution (i-Max II, INtRON Biotechnology) containing 1 μL of each primer (50 μM) and 20 ng genomic DNA, made up to 25 μL with water. Amplification was carried out on the GeneAmp PCR System (Applied Biosystems) at 94 °C for 5 min followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1.5 min, followed by a final 72 °C step for 7 min. PCR products were purified using NucleoFast 96 PCR plates (Machery-Nagel, Düren, Germany) and sequenced.
Isolation of total RNA and cDNA library construction
Total RNA from endophytes was isolated using the borax method. Mycelia were homogenized under liquid nitrogen using a mortar and pestle, incubated at 42 °C for 1 h in 15 mL borax buffer (0.2 M sodium tetraborate, 30 mM EGTA, 1 % (w/v) SDS, 1 % (w/v) deoxycholate, 1 % (v/v) Nonidet P-40, 2 % (w/v) polyvinylpyrolidone, 10 mM DDT, pH 9.0), supplemented with 1.2 mL 2 M KCl and stored on ice for 1 h. After centrifugation, RNA was selectively precipitated by adding 5 mL 8 M LiCl and storing at −20 °C overnight. The precipitate was washed three times with cold 2 M LiCl and resuspended in 2.8 mL TES buffer (50 mM Tri/HCl pH 5.7, 5 mM EDTA, 50 mM NaCl) supplemented with 1 M CsCl. This suspension was overlaid with 1.2 mL TES buffer supplemented with 5.7 M CsCl and the RNA was purified by density gradient ultracentrifugation at room temperature at 100,000 × g for 16 h. The RNA was dissolved in 500 μL TE buffer (10 mM Tris/HCl pH 8.0, 1 mM EDTA) and mRNA was isolated using the Qiagen Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany). A cDNA-RACE library was constructed using the Clontech Marathon cDNA Amplification Kit (Takara BIO Europe, Saint-Germain-en-Laye, France) according to the manufacturer’s instructions. Primers for the amplification of terpene synthase gene candidates are listed in Table S3.
Cloning, expression and functional testing of diterpene synthase 0021_TS_1762 and intron-1 splice variants of 0021_TS_1762
Synthetic diterpene synthase 1762 (Genskript, Hong Kong) was amplified using pUC57 as the template, and the product was transferred to the Escherichia coli expression vector pTrc-His2 using the pTrcHis2-TOPO® TA Expression Kit (Invitrogen, Karlsruhe, Germany). The gene was also amplified with primers including Gateway attachment sites allowing the gene to be introduced into the yeast expression vector pYES-Dest52 by homologous recombination. The protein was expressed in E. coli DH5α cells (New England Biolabs, Frankfurt, Germany) and Saccharomyces cerevisiae CEN-PK2-1 cells (EUROSCARF, Frankfurt, German) at 28 °C. Deletion variant 0021_TS_1762_del and intron1 random variants (primers listed in Table S3) were created by whole-plasmid PCR using pTrcHIS2-1762cosyn as the template with Herculase® II Fusion DNA Polymerase (Agilent Technologies, Karlsruhe, Germany) and the following temperature program: 95 °C for 3 min, followed by 30 cycles at 95 °C for 0.5 min, 58 °C for 0.5 min and 72 °C for 4 min, followed by a final step at 72 °C for 7 min. Crude protein extracts were prepared by disrupting the cells with glass beads. One volume of extract was used for in vitro testing with three volumes of assay buffer (100 mM Tris, 10 mM MgCl2, 5 mM β-mercaptoethanol, 50 μM substrate 3H-GGPP, 3H-FPP or 14C-IPP (+DMAPP), total volume 500 μL). Biotransformation reactions were incubated at 30 °C, overnight. After the addition of 500 μL saturated NaCl the reactions were extracted twice with the same volume of ethyl acetate. The extracts were concentrated in a nitrogen stream and analyzed by radio-TLC on silica plates (Merck, Darmstadt, Germany), which were developed with 9:1 cyclohexane:ethyl acetate or 3:1 pentane:diethyl ether. Products were detected using a radio-TLC Scanner RITA Star (Raytest, Straubenhardt, Germany).
Phage insert, ITS and whole genome sequencing
Phage inserts were sequenced using the Sanger method (Functional & Applied Genomics Group, Fraunhofer IME, Aachen, Germany) or shotgun sequencing (Eurofins MWG Operon, Ebersberg, Germany). ITS sequences were determined by Sanger sequencing (Functional & Applied Genomics Group, Fraunhofer IME). The EF0021 genome was sequenced using 454 technology by Seq-It GmbH, Kaiserslautern. The Taxomyces andreanae genome was sequenced by paired-end library sequencing (imagenes GmbH, Berlin, Germany). Each supplier also assembled the sequences they generated.
Sequences were analyzed using CLC Combined Workbench v3.6.1, Lasergene 7 Package, NCBI Blast and CloneManager Professional Suite 8. FGENESH was use for ORF and protein prediction (http://linux1.softberry.com/). Phylogenetic analysis was carried out using CLC Combined Workbench v3.6.1 with the protein sequences listed in Supplementary Data S3 and Table S4.