Biosynthetic Study of Cephalosporin P₁ Reveals a Multifunctional P450 Enzyme and a Site-Selective Acetyltransferase

Abstract

Fusidane-type antibiotics are a group of triterpenoid antibiotics. They include helvolic acid, fusidic acid, and cephalosporin P₁, among which fusidic acid has been used clinically. We have recently elucidated the biosynthesis of helvolic acid and fusidic acid, which share an early biosynthetic route involving six conserved enzymes. Here, we report two separate gene clusters for cephalosporin P₁ biosynthesis. One consists of the six conserved genes, and the other contains three genes encoding a P450 enzyme (CepB4), an acetyltransferase (CepD2), and a short-chain dehydrogenase/reductase (CepC2). Introduction of these three genes into <i>Aspergillus oryzae</i>, which harbors the six conserved genes, produced cephalosporin P₁. Stepwise introduction revealed that CepB4 not only catalyzes stereoselective dual oxidation of C6 and C7, but also monooxygenation of C6 or C7. This led to the generation of five new analogues. Using monohydroxylated products as substrates, we demonstrated that CepD2 specifically acetylates C6-OH, although both C6-OH and C7-OH acetylated analogues have been identified in nature.