Abstract

The newly discovered iron scavenger 7-hydroxytropolone (7-HT) is secreted by Pseudomonas donghuensis 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, paaZ (orf13) and ech, are synergistically involved in the biosynthesis of 7-HT in P. donghuensis HYS. Firstly, gene knockout and a sole carbon experiment indicated that the genes orf17-21 (paaEDCBA) and orf26 (paaG) were involved in the biosynthesis of 7-HT and participated in the PAA catabolon pathway in P. donghuensis HYS; these genes were arranged in gene cluster 2 in P. donghuensis HYS. Interestingly, ORF13 was a homologous protein of PaaZ, but orf13 (paaZ) was not essential for the biosynthesis of 7-HT in P. donghuensis HYS. A genome-wide BLASTP search, including gene knockout, complemented assays, and site mutation, showed that the gene ech homologous to the ECH domain of orf13 (paaZ) is essential for the biosynthesis of 7-HT. Three key conserved residues of ech (Asp39, His44, and Gly62) were identified in P. donghuensis HYS. Furthermore, orf13 (paaZ) could not complement the role of ech in the production of 7-HT, and the single carbon experiment indicated that paaZ mainly participates in PAA catabolism. Overall, this study reveals a natural association between PAA catabolon and the biosynthesis of 7-HT in P. donghuensis HYS. These two genes have a synergistic effect and different functions: paaZ is mainly involved in the degradation of PAA, while ech is mainly related to the biosynthesis of 7-HT in P. donghuensis HYS. These findings complement our understanding of the mechanism of the biosynthesis of 7-HT in the genus Pseudomonas.

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