To ). Detailed NMR analysis confirmed the structure as 2, herein named preburnettiene B, as containing a free carboxylic acid group (5-CO2H) in place of the methyl ester in 1 (Fig. , Table , Figs. S14-S17 †). Additionally, we observed that after 14 days of large-scale cultivation, 2 was completely converted to 1 (Fig, ). Overall, these results demonstrate that BueF is responsible for methylation of the 5-CO2H group on 2 and is required for the biosynthesis of 1 (Fig. ).