<jats:title>Abstract</jats:title><jats:p>To isolate a key polyketide biosynthetic intermediate for the 16‐membered macrolide FD‐891 (<jats:bold>1</jats:bold>), we inactivated two biosynthetic genes coding for post‐polyketide synthase (PKS) modification enzymes: a methyltransferase (GfsG) and a cytochrome P450 (GfsF). Consequently, FD‐892 (<jats:bold>2</jats:bold>), which lacks the epoxide moiety at C8–C9, the hydroxy group at C10, and the <jats:italic>O</jats:italic>‐methyl group at O‐25 of FD‐891, was isolated from the <jats:italic>gfsF</jats:italic>/<jats:italic>gfsG</jats:italic> double‐knockout mutant. In addition, 25‐<jats:italic>O</jats:italic>‐methyl‐FD‐892 (<jats:bold>3</jats:bold>) and 25‐<jats:italic>O</jats:italic>‐demethyl‐FD‐891 (<jats:bold>4</jats:bold>) were isolated from the <jats:italic>gfsF</jats:italic> and <jats:italic>gfsG</jats:italic> mutants, respectively. We also confirmed that GfsG efficiently catalyzes the methylation of <jats:bold>2</jats:bold> and <jats:bold>4</jats:bold> in vitro. Further, GfsF catalyzed the epoxidation of the double bond at C8‐C9 of <jats:bold>2</jats:bold> and <jats:bold>3</jats:bold> and subsequent hydroxylation at C10, to afford <jats:bold>4</jats:bold> and <jats:bold>1</jats:bold>, respectively. These results suggest that a parallel post‐PKS modification mechanism is involved in FD‐891 biosynthesis.