To elucidate the intermediates in the biosynthetic pathway to 1, we evaluated truncated versions of the bue cluster by heterologous expression in A. nidulans (Fig. ). We first hypothesised that the methyltransferase bueF would be responsible for the last step of biosynthesis. When the vector encoding the methyltransferase bueF was not transformed, we observed high production of compound 2 with a UV-vis spectrum matching to 1 and a mass ion m/z of 523 [M -H] - (Figs. and). The difference in mass of 14 Da was attributed to the presence of a free carboxylic acid in 2 compared to the methyl ester in 1. We then cloned a truncated version of the cluster also omitting the gene encoding the cytochrome p450 BueE. In the strains expressing bueA/B/C/D/G/R, we observed peak 3 at high retention time (14.24 min) with a UV-vis spectrum similar to 1 and m/z 463 [M -H] -(Fig. and S29 †).