Identification of the <i>p-</i>coumaric acid biosynthetic gene cluster in <i>Kutzneria albida</i>: insights into the diazotization-dependent deamination pathway

Abstract

Recently, we identified the biosynthetic gene cluster of avenalumic acid (<i>ava</i> cluster) and revealed its entire biosynthetic pathway, resulting in the discovery of a diazotization-dependent deamination pathway. Genome database analysis revealed the presence of more than 100 <i>ava</i> cluster-related biosynthetic gene clusters (BGCs) in actinomycetes; however, their functions remained unclear. In this study, we focused on an <i>ava</i> cluster-related BGC in <i>Kutzneria albida</i> (<i>cma</i> cluster), and revealed that it is responsible for <i>p-</i>coumaric acid biosynthesis by heterologous expression of the <i>cma</i> cluster and in vitro enzyme assays using recombinant Cma proteins. The ATP-dependent diazotase CmaA6 catalyzed the diazotization of both 3-aminocoumaric acid and 3-aminoavenalumic acid using nitrous acid in vitro. In addition, the high efficiency of the CmaA6 reaction enabled us to perform a kinetic analysis of AvaA7, which confirmed that AvaA7 catalyzes the denitrification of 3-diazoavenalumic acid in avenalumic acid biosynthesis. This study deepened our understanding of the highly reducing type II polyketide synthase system as well as the diazotization-dependent deamination pathway for the production of avenalumic acid or <i>p-</i>coumaric acid.