Co-factor independent oxidases ncnN and actVA-3 are involved in the dimerization of benzoisochromanequinone antibiotics in naphthocyclinone and actinorhodin biosynthesis

Abstract

<p>Streptomyces produce complex bioactive secondary metabolites with remarkable chemical diversity. Benzoisochromanequinone polyketides actinorhodin and naphthocyclinone are formed through dimerization of half-molecules via single or double carbon-carbon bonds, respectively. Here we sequenced the genome of <i>S. arenae</i> DSM40737 to identify the naphthocyclinone gene cluster and established heterologous production in <i>S. albus</i> J1074 by utilizing direct cluster capture techniques. Comparative sequence analysis uncovered <i>ncnN</i> and <i>ncnM</i> gene products as putative enzymes responsible for dimerization. Inactivation of <i>ncnN</i> that is homologous to atypical co-factor independent oxidases resulted in the accumulation of fogacin, which is likely a reduced shunt product of the true substrate for naphthocyclinone dimerization. In agreement, inactivation of the homologous <i>act</i>VA-3 in <i>S. coelicolor</i> M145 also led to significantly reduced production of actinorhodin. Previous work has identified the NAD(P)H-dependent reductase ActVA-4 as the key enzyme in actinorhodin dimerization, but surprisingly inactivation of the homologous <i>ncnM</i> did not abolish naphthocyclinone formation and the mutation may have been complemented by an endogenous gene product. Our data suggests that dimerization of benzoisochromanequinone polyketides require two-component reductase-oxidase systems.<br></p>