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

Abstract

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