Biosynthesis of Benzohydroxamic Acid in Streptomyces angustmyceticus

Benzohydroxamic acid (BHA) is an important hydroxamate scaffold with potential applications in medicinal chemistry and crop protection, but it has not previously been established as a natural product. Here, we report the discovery and biosynthetic characterization of BHA from Streptomyces angustmyceticus. A conserved TsnB-like gene cassette comprising SaAmOx-AS, SaAmOx, and SaHAT was identified by genome mining, indicating a potential capacity for hydroxamate formation and transfer. BHA was detected as a fermentation product of S. angustmyceticus and structurally confirmed by comparison with an authentic standard, compound purification, and NMR spectroscopic analysis. Benzoic acid was established as the aromatic precursor of BHA through deuterium-labeling experiments with benzaldehyde and cinnamic acid. SaHAT was identified as the key hydroxamate-transfer enzyme that directly converts benzoic acid, rather than benzoyl-CoA, to BHA using glutamyl hydroxamate as the hydroxamate donor in an ATP- and Mg²⁺-dependent reaction. BHA production was reconstituted in Escherichia coli by heterologous coexpression of SaAmOx-AS, SaAmOx, and SaHAT, confirming that these three enzymes form a minimal biosynthetic module. This work establishes BHA as a microbial natural product and provides a foundation for engineering hydroxamate-containing compounds.