# Role of a FAD-dependent monooxygenase in diazo group functionalization of kinamycin in Streptomyces ambofaciens

## TL;DR

Scientists discovered a key enzyme involved in making a cancer-fighting compound in a soil bacterium.

## Contribution

A FAD-dependent monooxygenase essential for diazo group formation in kinamycin biosynthesis is identified.

## Key findings

- A FAD-dependent monooxygenase is essential for diazo functionalization in kinamycin biosynthesis.
- Absence of the enzyme leads to accumulation of stealthin C as a pathway shunt product.
- The enzyme's gene location defines a 72 kb kinamycin biosynthetic gene cluster.

## Abstract

Kinamycin biosynthesis is a complex process that has been extensively studied over the years, yet specific enzymatic steps continue to be unveiled. A diazo group present in the molecule is responsible for the promising antitumour activity of kinamycins, but its installation in the specific strain Streptomyces ambofaciens has yet to be characterized. In this study, we explore the diazo functionalization of kinamycin in this strain. A FAD-dependent monooxygenase is identified, which is essential for kinamycin biosynthesis. In its absence, stealthin C accumulates instead, likely as a pathway shunt product. Furthermore, as a result of the position of the gene encoding this monooxygenase, named alp2F, we also propose new boundaries of the kinamycin biosynthetic gene cluster, resulting in a large cluster spanning over 72 kb. This work paves the way for the continued understanding of the biosynthetic steps that are characteristic of diazo-containing natural products and provides new biocatalysts for molecular engineering and accelerates bioactive compounds production.