Identification of a Novel D-alanine–D-alanine Ligase from Nocardioides kribbensis for Efficient In Vivo Biosynthesis of Shinorine in Engineered Escherichia coli

Abstract Shinorine, a UV-absorbing mycosporine-like amino acid (MAA), is biosynthesized from sedoheptulose 7-phosphate via a conserved gene cluster (mysA, mysB, mysC, and mysD). The final step, catalyzed by MysD, a homolog of D-alanine–D-alanine ligase (DDL), converts mycosporine-glycine to shinorine. In this study, a novel MysD from Nocardioides kribbensis was expressed, purified, and biochemically characterized. The enzyme showed optimal activity at pH 8.0 and 40 °C, with obvious enhancement by Mg2+ and K+. Kinetic analysis revealed Michaelis constants (Km) of 78.7 μM for ATP, 84 μM for the first D-alanine, and 785 μM for the second D-alanine, with a catalytic efficiency (kcat/Km) of 433 M–1 s–1. Chromosomal integration of this gene into a mycosporine-glycine-producing engineered strain enabled efficient shinorine biosynthesis. Through strain engineering combined with fed-batch fermentation, shinorine titers reached 243.36 mg/L in shake-flask culture and 1.96 g/L in fed-batch fermentation, demonstrating the potential for scalable microbial production of this natural sunscreen compound.