2 and Suppl. Data S2). LC/MS/MS analysis confirmed the initial results obtained with CIEIA for EF0001, but Taxol, #GANT61 research buy randurls[1|1|,|CHEM1|]# baccatin III and 10-deacetylbaccatin III were not detected by CIEIA or LC/MS/MS in any of the other species. Fig. 2 LC/MS/MS-multi-reaction monitoring (MRM) analysis of an organic extract

from the Taxus endophyte EF0021. a LC/MS/MS-MRM chromatogram of 10-deacetylbaccatin III (10-DABIII, authentic standard (Idena, Milano, Italy), dissolved in methanol at a concentration of 1 mg/mL, injection volume 10 μL) eluting from the HPLC column at 4.72 min. The insert shows the three monitored ion transitions (m/z = 76.2, 120.8 and 391.2) of the 10-DABIII parent ion (m/z = 543.2) (M-H). b LC/MS/MS-MRM chromatogram with the observed mass pattern (shown in insert) at 4.72 min obtained with the organic extract of Taxus endophyte EF0021 Without delay (assuming potential genetic instability in the fungi), we extracted genomic DNA from EF0001 and EF0021. To avoid potential contamination leading to PCR artifacts, we established genomic phage libraries for both species

and used conventional hybridization as the screening method. We used three probes specific for Taxol biosynthesis: taxadiene synthase (Wildung and Croteau 1996), taxane-5α-hydroxylase (Jennewein et al. 2004a), and taxane-13α-hydroxylase (Jennewein et al. 2001). For EF0001, we screened a total of 300,000 phage plaques (average insert size, 23 kb) corresponding to ~6,900 Mb of endophyte genomic sequence. Assuming an average fungal genome size of 50 Mb, this strategy achieved >130-fold genome coverage. For EF0021, mTOR inhibitor we screened a total of 40,000 phage plaques, corresponding to 920 Mb of genomic sequence and 18-fold genome coverage. Several potential positive Telomerase inserts were sequenced, but none of them

corresponded to known Taxus spp. genes involved in taxane biosynthesis. Given that we were unable to identify taxane-related genomic sequences in EF0001 and ER0021, we constructed a T. andreanae genomic phage library and screened 162,000 phage plaques (average insert size 20.3 kb, corresponding to 3,300 Mb of genomic sequence and 66-fold genome coverage) using the same probes as above and did not identify any positive clones. Our failure to identify fungal genomic sequence related to known taxane-specific sequences from yew trees led us to conclude that taxane biosynthesis in endophytes may have evolved independently, as is the case for gibberellins, whose biosynthesis pathway differs between microbes and plants (Tudzynski and Hölter 1998; Bömke and Tudzynski 2009). To further examine the potential for independent taxane biosynthesis by endophytes, we sequenced the EF0021 genome using a shotgun sequencing approach, yielding 2,234,101 sequence reads with an average length of 390 bp. Sequence alignment of the raw data achieved 98.55 % aligned reads and 2,623 contigs covering 44.45 Mb of genomic DNA, corresponding to an estimated genome size of 45.9 Mb.