1 The cluster encodes proteins showing similarity to a hybrid modular PKS and to

several enzymes involved in post PKS modifications pointing to a highly functionalised molecule. To discover metabolites that correspond to the presence of this orphan PKS gene cluster, we performed a systematic analysis of the secondary metabolome of the B. gladioli strain. Interestingly, besides bongkrekic acid and toxoflavin, no other secondary metabolites were found even though various culture conditions were tested. This indicates that the PKS gene cluster is not expressed under common laboratory culture conditions and is very likely only induced upon a certain trigger. One way to induce the expression of such silent genes is to mimic the natural habitat of an organism, i.e. to simulate a scenario potentially occurring in the field.[35, 43, 45-47] Therefore we hypothesised Temsirolimus that either culture conditions mimicking the food fermentation process or the presence of the associated fungus R. microsporus might provide the required DAPT in vitro cue to activate the silent or down-regulated genes. To prove this hypothesis, we first cultured B. gladioli as a stationary culture on liquid and solid media thus reducing the oxygen supply as it is very likely the case during the fermentation

of tempe[48] and monitored secondary metabolite formation by LC-MS. Indeed, we noticed the formation of a number of related compounds that were previously not observed (Fig. 2). MS and UV analyses and dereplication employing natural product databases pointed many to a potential identity with enacyloxins. These compounds were previously isolated from Frateuria sp. and Burkholderia ambifaria.[49-53] To prove that the induced products are identical with enacyloxins, we isolated the derivatives from a large-scale culture by a combination of different chromatographic techniques and elucidated their structures by 1D and 2D NMR analyses. In total, we yielded four different compounds. For compound 3, a

molecular formula of C33H45NO11Cl2 was deduced from HRESI-MS. The 1H and 13C NMR spectra were in good agreement with the published data of enacyloxin IIa.[53] 2D NMR analyses corroborated the proposed structure. Compound 4 was found to be identical to iso-enacyloxin IIa (Fig. 1a).[53] The molecular composition of compound 5 was determined to be C33H48NO11Cl indicating the presence of a mono-halogenated derivative. In contrast to compounds 3 and 4, the 13C NMR spectrum did not display a signal of a ketone, but an additional oxymethine as well as another methylene function instead (Table 1). Analyses of the H,H-COSY and the HMBC couplings identified compound 5 as enacyloxin IIIa. Compound 6 proved to be the corresponding isomer of 5 and thus represents a novel metabolite.