Decoding Specificity of Cyanobacterial MysDs in Mycosporine-like Amino Acid Biosynthesis through Heterologous Expression in <i>Saccharomyces cerevisiae</i>

Mycosporine-like amino acids (MAAs) are potent natural UV-protectants, but their industrial production is hindered by efficiency and sustainability issues in large-scale extraction of native hosts. Heterologous biosynthesis in chassis organisms provides a promising alternative route, although the substrate promiscuity of the ATP-grasp ligase MysD complicates the biosynthesis of specific MAAs. In this study, we developed a <i>Saccharomyces cerevisiae</i> strain with enhanced capacity of producing mycosporine-glycine (MG). This strain serves as an efficient MysD expression platform that converts MG into shinorine and porphyra-334. Through structural modeling, site-directed mutagenesis, and mutant characterization, we identified two residues on the omega-loop of MysD involved in determining product specificity. We further characterized the product specificity of 20 MysDs from diverse cyanobacterial lineages and confirmed the residue pattern-product specificity correlation. Our findings provide guidance for screening, selecting, and designing novel MysDs for industrial-scale MAA production through heterologous expression.