Efficient Production of Shinorine and Mycosporine-Glycine-Alanine in Using Natural and Engineered Nonribosomal Peptide Synthetases.

Mycosporine-like amino acids (MAAs) are ultraviolet-absorbing metabolites with recognized photoprotective and pharmacological benefits, predominantly found in marine organisms. Due to the inefficiency of extraction from natural sources, microbial biosynthesis using heterologous hosts is an attractive alternative. Disubstituted MAAs, such as shinorine, porphyra-334, and mycosporine-2-glycine, are synthesized by conjugating serine, threonine, or glycine to mycosporine-glycine (MG), a reaction catalyzed by either a d-Ala-d-Ala ligase homolog (MysD) or a nonribosomal peptide synthetase (MysE). While MysD enzymes often yield diverse byproducts due to substrate promiscuity, MysE demonstrates higher substrate specificity; however, only a serine-specific MysE has been biochemically characterized. In this study, we enhanced shinorine production in by expressing (). Functional expression required coexpression of a phosphopantetheinyl transferase (PPTase), which was absent in but native to . The PPTase supported the MysE activity in both hosts. Furthermore, we engineered the MysE adenylation domain to alter specificity from serine to alanine, enabling biosynthesis of MG-alanine, a rare MAA previously detected only as a minor MysD byproduct. These findings demonstrate the utility of MysE engineering for expanding MAA diversity and advancing the sustainable microbial production of novel sunscreen compounds.