The Post‐Polyketide Synthase Modification Mechanism in Hitachimycin Biosynthesis

Abstract

Hitachimycin is a bicyclic macrolactam antibiotic with (S)‐β‐phenylalanine (β‐Phe) at the starter position of the polyketide skeleton. While the enzymes that recognize β‐amino acids, modify the aminoacyl groups, and transfer the resultant dipeptide groups to the acyl carrier protein domains of polyketide synthases (PKSs) have been studied extensively, the post‐PKS modification mechanism responsible for constructing the unique bicyclic structure of hitachimycin remains elusive. In this study, we first inactivated six genes encoding putative post‐PKS modification enzymes, namely hitM1 to hitM6, in Streptomyces scabrisporus to determine their involvement in hitachimycin biosynthesis. The ΔhitM4 strain accumulated an all‐trans‐2,4,6,8,18‐pentaene macrolactam, which was confirmed as a true intermediate in hitachimycin biosynthesis by cellular feeding experiments, and appears to be the initial intermediate in the post‐PKS modification pathway. The ΔhitM1 strain accumulated 10‐O‐demethyl‐10‐oxohitachimycin (M1‐A). In enzymatic experiments, M1‐A was reduced by the NAD(P)H‐dependent reductase HitM1 in the presence of NADPH. The product of the reaction catalyzed by HitM1 was converted to hitachimycin by the methyltransferase HitM6. We thus propose a plausible post‐PKS modification mechanism for the biosynthesis of hitachimycin.