Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin

Abstract

Natural products containing nitrogen-nitrogen (N-N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous acid-dependent N-N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown. Herein, we focused on spinamycin, a hydrazide-containing aryl polyene produced by <i>Streptomyces albospinus</i> JCM3399. In the <i>S. albospinus</i> genome, we discovered a putative spinamycin biosynthetic gene (<i>spi</i>) cluster containing genes that encode a type II polyketide synthase and genes for the secondary metabolism-specific nitrous acid biosynthesis pathway. A gene inactivation experiment showed that this cluster was responsible for spinamycin biosynthesis. A feeding experiment using stable isotope-labeled sodium nitrite and analysis of nitrous acid-synthesizing enzymes <i>in vitro</i> strongly indicated that one of the nitrogen atoms of the hydrazide group was derived from nitrous acid. <i>In vitro</i> substrate specificity analysis of SpiA3, which is responsible for loading a starter substrate onto polyketide synthase, indicated that N-N bond formation occurs after starter substrate loading. <i>In vitro</i> analysis showed that the AMP-dependent ligase SpiA7 catalyzes the diazotization of an amino group on a benzene ring without a hydroxy group, resulting in a highly reactive diazo intermediate, which may be the key step in hydrazide group formation. Therefore, we propose the overall biosynthetic pathway of spinamycin. This study expands our knowledge of N-N bond formation in microbial secondary metabolism.