Characterization of a silent sesquiterpenoid biosynthetic pathway in Streptomyces avermitilis controlling epi‐isozizaene albaflavenone biosynthesis and isolation of a new oxidized epi‐isozizaene metabolite

The genome-sequenced, Gram-positive bacterium Streptomyces avermitilis harbours an orthologue (SAV_3032) of the previously identified epi-isozizaene synthase (SCO5222) in Streptomyces coelicolor A3(2). The sav3032 is translationally coupled with the downstream sav3031 gene encoding the cytochrome P450 CYP170A2 analogous to SCO5223 (CYP170A1) of S. coelicolor A3(2), which exhibits a similar translation coupling. Streptomyces avermitilis did not produce epi-isozizaene or any of its oxidized derivatives, albaflavenols and albaflavenone, under in any culture conditions examined. Nonetheless, recombinant SAV_3032 protein expressed in Escherichia coli catalysed the Mg²+-dependent cyclization of farnesyl diphosphate to epi-isozizaene. To effect the production of epi-isozizaene in S. avermitilis, the sav3032 gene was cloned and placed under control of a copy of the native S. avermitilis promoter rpsJp (sav4925). The derived expression construct was introduced by transformation into a large-deletion mutant of S. avermitilis SUKA16 and the resulting transformants accumulated epi-isozizaene. The previously characterized oxidized epi-isozizaene metabolites (4R)- and (4S)-albaflavenols and albaflavenone, as well as a previously undescribed doubly oxidized epi-isozizaene derivative were isolated from cultures of S. avermitilis SUKA16 transformants in which sav3032 was coexpressed with the P450-encoding sav3031. This new metabolite was identified as 4β,5β-epoxy-2-epi-zizaan-6β-ol which is most likely formed by oxidation of (4S)-albaflavenol.