The Gene of the Phenylacetic Acid (PAA) Catabolic Pathway Branching Point and Outside the PAA Catabolon Gene Cluster Are Synergistically Involved in the Biosynthesis of the Iron Scavenger 7-Hydroxytropolone in HYS

The newly discovered iron scavenger 7-hydroxytropolone (7-HT) is secreted by HYS. In addition to possessing an iron-chelating ability, 7-HT has various other biological activities. However, 7-HT's biosynthetic pathway remains unclear. This study was the first to report that the phenylacetic acid (PAA) catabolon genes in cluster 2 are involved in the biosynthesis of 7-HT and that two genes, () and , are synergistically involved in the biosynthesis of 7-HT in HYS. Firstly, gene knockout and a sole carbon experiment indicated that the genes () and () were involved in the biosynthesis of 7-HT and participated in the PAA catabolon pathway in HYS; these genes were arranged in gene cluster 2 in HYS. Interestingly, ORF13 was a homologous protein of PaaZ, but () was not essential for the biosynthesis of 7-HT in HYS. A genome-wide BLASTP search, including gene knockout, complemented assays, and site mutation, showed that the gene homologous to the ECH domain of () is essential for the biosynthesis of 7-HT. Three key conserved residues of (Asp39, His44, and Gly62) were identified in HYS. Furthermore, () could not complement the role of in the production of 7-HT, and the single carbon experiment indicated that mainly participates in PAA catabolism. Overall, this study reveals a natural association between PAA catabolon and the biosynthesis of 7-HT in HYS. These two genes have a synergistic effect and different functions: is mainly involved in the degradation of PAA, while is mainly related to the biosynthesis of 7-HT in HYS. These findings complement our understanding of the mechanism of the biosynthesis of 7-HT in the genus .