Investigations into the biosynthesis of stieleriacines and related N-acyl tyrosines by comparative genomics, knock-out studies and total synthesis of epi-stieleriacine C.

N-acyl tyrosines, a prominent class of N-acyl amino acid biomolecules, are produced by selected species in at least three bacterial phyla: Pseudomonadota, Actinomycetota and Planctomycetota. Long-chain N-acyl tyrosines with a characteristic 2,3-dehydrotyrosine core structure and additional taxon-specific chemical modifications were previously reported under the names thalassotalic acids, kyonggic acids and stieleriacines. However, the underlying pathway for their biosynthesis in the different bacterial taxa remains largely unexplored. Here, we focused on the identification of biosynthetic enzymes in the two known stieleriacine-producing planctomycetal strains of the eponymous genus Stieleria. Comparative genome analyses of stieleriacine-, thalassotalic acid- and kyonggic acid producers suggest a common pathway for N-acyl dehydrotyrosine biosynthesis based on conserved genes encoding a putative adenylyltransferase/cyclase, nitroreductase and the hallmark protein N-acyl amino acid synthase (NasY). The targeted deletion of three predicted nasY genes in Stieleria neptunia indicates that one of the three encoded enzymes predominantly produces stieleriacines. We also confirmed the absolute structure of stieleriacine C by synthesis of its epimer and structural derivatives, which serve as the basis for the future investigation of the biological function of N-acyl tyrosines.