[1S05m] 13. Microbial Engineering for Natural Products Synthesis - Part 1

11:15 AM - 11:30 AM JST(2:15 AM - 2:30 AM UTC)

[1MENP-04] Diversity of secondary metabolite biosynthetic pathways with diazo group synthesis in actinobacteria

○Yohei Katsuyama^1,2 (1. The University of Tokyo (Japan), 2. Collaborative Research Institute for Innovative Microbiology (Japan))

Keywords:

biosynthesis,actinobacteria,diazo,enzyme,secondary metabolism

Actinomycetes are prolific producers of useful natural products. Interestingly, some of them are known to produce diazo-containing natural products, which frequently exhibit potent antimicrobial and anticancer properties driven by the high reactivity of the diazo group. Although their biosynthetic origins remained elusive for decades, recent studies have provided significant advance. Through the study of cremeomycin, our group discovered that a specialized nitrous acid biosynthetic pathway dedicated to secondary metabolism in actinomycetes is used for biosynthesis of the diazo group. Leveraging genome mining for these pathway genes, we identified that this pathway is also used for the diazo group biosynthesis of compounds other than cremeomycin. In addition, we discovered biosynthetic pathways where diazo-containing molecules serve as an intermediate. In the biosynthesis of avenalumic acid and p-coumaric acid, diazo group synthesis was used to remove the aromatic amine. In contrast, it was used to synthesize hydrazide moiety in spinamycin biosynthesis. Furthermore, we discovered that it is also used for the biosynthesis of the diaminophenol moiety in several secondary metabolites including nybomycin. By comparing the secondary metabolite biosynthetic gene clusters which presumably includes diaminophenol moiety, we discovered 9 genes conserved among these gene clusters. By the analysis of these genes, we showed that the four of these genes are responsible for biosynthesis of 3-hydroxyanthranilic acid, and three genes are responsible for the aromatic amination of 3-hydroxyanthranilic acid using nitrous acid to synthesize 2,4-diamino-3-hydroxybenzoic acid via diazotization. These findings exemplify the functional diversity of diazo groups in the biosynthesis of secondary metabolites. Furthermore, we investigated the mechanisms of various diazotases within these pathways. Notably, we determined the structure of CmaA6, which diazotizes 3-amino-p-coumaric acid, using X-ray crystallography and cryo-EM. The structure of CmaA6 binding to AMP, allowed us to identify the recognition sites for nitrous acid and aromatic amines. Structural information combined with site-directed mutagenesis, kinetic studies, and computational modeling, enabled us to propose a reaction mechanism for CmaA6.

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