, 1987a, b) or to Saccharomyces cerevisiae expressing norA or ordA after induction with

galactose (Yu et al., 1998). Following a 4-h incubation, metabolites were extracted into methylene chloride and aliquots were examined by TLC. blast searches (tblastx and blastp) were performed against the sequenced fungal genome datasets in Pubmed (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), the Broad Institute fungal database (http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html), and the A. flavus genomic sequence (http://www.aspergillusflavus.org/genomics). The cut-off for matches 17-AAG was E−30. Transformation of A. flavus AF13ΔniaD with the linearized norA knockout vector (Fig. 2a) yielded approximately 60 colonies, three of which had

slightly darker orange mycelia when regrown on PDA plates. The three darker orange transformants click here were confirmed to be double crossover norA disruptants by PCR (Fig. 2b). A 1.5-kb PCR band was obtained for intact norA in the AF13 control strain and an 8 kb product for the positive ΔnorA transformants (Fig. 2b). The latter product is consistent with the size expected with the 7 kb niaD selection marker inserted into the norA gene. Only acetone extracts of the norA knockout cultures and cultures transformed with the selection marker were examined by liquid chromatography combined with mass spectrometry (LC/MS; Fig. 3 and Table 1). A metabolite eluted after AFB1 (14.1 min compared with 13.7 min) and exhibited a blue-shifted (λmax=332 nm) chromophore compared with that of AFB1 (λmax=362 nm). This less polar compound was identified as deoxyAFB1 by its positive ion mass spectrum (M+H=297; deoxyAFB1, M=296 Da) and having a retention time and UV-visible chromophore identical to that of deoxyAFB1 prepared by established synthetic methods (Hsia & Chu, 1977). The LC data showed that deoxyAFB1 accumulated in at least 20-fold greater

amounts in the norA knockout strain than in the selection marker-only transformed strain (Fig. 3). Comparison of other metabolites in the acetone extracts of an AF13ΔnorA clone (#15) and the AF13 control with natural or synthetic standards by UV-visible spectrophotometry and positive ion LC/MS confirmed the presence of OMST (15.9 min), HOMST (12.4 min, M+H=355, M=354), and AFB1 (13.8 min) (Table 1). Montelukast Sodium The metabolites shared identical LC retention times, UV-visible chromophores, and mass spectra with their respective standard. Several unknown compounds were also observed in extracts of fungi with both mutant and intact norA. One exhibited a chromophore (λmax=318 nm, shoulder at 360 nm; M+H=371, M=370) similar to those of OMST and HOMST, suggesting that it could be a related intermediate in the pathway. Two unknown compounds eluting at 10.9 and 13.0 min with the same mass (M+H=329, M=328) were found in extracts from control and norA mutant fungi. One of them eluted at 10.9 and 13.0 min, and exhibited a chromophore similar to that of AFB1 (λmax=360 nm).