It has previously been shown that orsA (AN7909) is involved in the formation of orsellinic acid (2), lecanoric acid (15), the two colored compounds F-9775A (16) and F-9775B (17), orcinol, diorcinol, gerfeldin and deoxy-gerfeldin. (Schroeckh et al., 2009; Sanchez et al., 2010). Our analysis confirms the link between orsellinic acid, lecanoric acid, diorcinol, F-9775A, F-9775B to orsA as these compounds are missing in the orsAΔ strain. However, we have not been able to detect the gerfeldins in any of our strains, and apparently our conditions favor violaceol and not gerfeldin

formation. The violaceols are formed by dimerization of two C7 monomers of 5-methylbenzene-1,2,3-triol, a compound that we could tentatively detect as [M-H]− at m/z 139 in cultivation find more extracts. The C7 backbone of 5-methylbenzene-1,2,3-triol, Alectinib manufacturer may conceivably be formed by decarboxylation of a C8 aldol intermediate as suggested by Turner 40 years ago (Turner, 1971) (Fig. 5). This C8 intermediate also serves as a branch point towards orsellinic acid. Interestingly, the same compounds that disappear in the orsAΔ strain also disappear in AN7903Δ, a strain missing a PKS gene separated from orsA by only ∼20 kb (Fig. 4). This result does not contradict the original assignment of orsA as the PKS gene responsible for production of orsellinic acid. Although the enzymes encoded by the two genes are predicted

to share many of the same functional domains, AN7903 is larger by around 500 amino acid residues and contains a methyl-transferase domain, which is not required for orsellinic acid production. Moreover, we note that Schroeckh et al. (2009) observed that both AN7903 and orsA were upregulated when orsellinic acid was induced by co-cultivation with Streptomyces hygroscopicus,

indicating cross-talk between the two clusters. Surprisingly, what appear to be trace amounts of orsellinic acid can be detected as m/z 167 [M-H]− in both the AN7903Δ and the orsAΔ strains (Fig. 4). The source of this residual orsellinic acid remains elusive, but it could possibly stem C59 from unmethylated byproducts from the PKS, AN8383, that produces 3,5-dimethylorsellinic acid, see below. Interestingly, production of austinol (18) and dehydroaustinol (19) was observed in the reference strain on several media (Fig. 1). Despite the fact that the production of these compounds is known from A. nidulans (Szewczyk et al., 2008), they have not yet been assigned to a specific gene. Only the AN8383Δ strain failed to produce the two austinols on all the media, which triggered austinol production in the reference strain (Fig. 6a). This, phenotype could be rescued by inserting the structural gene of AN8383 under the control of the gdpA promoter into an ectopic locus, IS1 (Hansen et al., 2011) (Fig. 6a). Moreover, a point mutant strain AN8383-S1660A also failed to produce austinols on these six media (Fig. 6a).