Function of Cytochrome P450 Enzymes MycCI and MycG in Micromonospora griseorubida, a Producer of the Macrolide Antibiotic Mycinamicin

<jats:title>ABSTRACT</jats:title> <jats:p> The cytochrome P450 enzymes MycCI and MycG are encoded within the mycinamicin biosynthetic gene cluster and are involved in the biosynthesis of mycinamicin II (a 16-membered macrolide antibiotic produced by <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Micromonospora griseorubida</jats:named-content> ). Based on recent enzymatic studies, MycCI is characterized as the C-21 methyl hydroxylase of mycinamicin VIII, while MycG is designated multifunctional P450, which catalyzes hydroxylation and also epoxidation at C-14 and C-12/13 on the macrolactone ring of mycinamicin. Here, we confirm the functions of MycCI and MycG in <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">M. griseorubida</jats:named-content> . Protomycinolide IV and mycinamicin VIII accumulated in the culture broth of the <jats:italic>mycCI</jats:italic> disruption mutant; moreover, the <jats:italic>mycCI</jats:italic> gene fragment complemented the production of mycinamicin I and mycinamicin II, which are produced as major mycinamicins by the wild strain <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">M. griseorubida</jats:named-content> A11725. The <jats:italic>mycG</jats:italic> disruption mutant did not produce mycinamicin I and mycinamicin II; however, mycinamicin IV accumulated in the culture broth. The <jats:italic>mycG</jats:italic> gene was located immediately downstream of the self-resistance gene <jats:italic>myrB</jats:italic> . The <jats:italic>mycG</jats:italic> gene under the control of <jats:italic>mycGp</jats:italic> complemented the production of mycinamicin I and mycinamicin II. Furthermore, the amount of mycinamicin II produced by the strain complemented with the <jats:italic>mycG</jats:italic> gene under the control of <jats:italic>myrBp</jats:italic> was approximately 2-fold higher than that produced by the wild strain. In <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">M. griseorubida</jats:named-content> , MycG recognized mycinamicin IV, mycinamicin V, and also mycinamicin III as the substrates. Moreover, it catalyzed hydroxylation and also epoxidation at C-14 and C-12/13 on these intermediates. However, C-14 on mycinamicin I was not hydroxylated. </jats:p>