-m/z 463.3239 indicative of a molecular formula C31H44O3 (∆mmu 2.1) containing four oxygen atoms fewer and four hydrogen atoms greater than 2 (Fig. ). The 1 H and C NMR data for 3 were very similar to those for 2 (Figs. S20 and S21 and Table †), with the only significant difference being the absence of signals for the carboxylic acid groups at C-1 and on C-5, and the presence of signals attributable to two additional methyl groups (δH 1.57, d; δC 13.6 and δH 1.46, s; δC 22.5). A COSY correlation between δH 1.57 and H-2 confirmed the first methyl group to be at C-1, while HMBC correlations from δH 1.46 to C-4, C-5 and C-6 confirmed the second methyl group to be on C-5 (Figs. S22-24 †). Thus, the structure of intermediate 3 was assigned as shown in Fig. . This compound is herein named preburnettiene A. We propose that the cytochrome p450 BueE catalyses regiospecific multi-oxidations at C-1 and 5-Me on 3 to the dicarboxylic acids on 2 (Fig. ). Thus, the activity of bueE targeting the branched chains differs to FSL4 from the fusarielin BGC which is responsible of oxidising the decalin core. The triple oxidation of a methyl group to produce a carboxylic acid has been previously observed to be catalysed by a single cytochrome P450 enzyme in fungi. However, BueE has the particularity of catalysing the reaction in two terminal methyl groups.