Engineering mandelate dehydrogenase for the efficient biosynthesis of salvianic acid A

Salvianic acid A (SAA), a bioactive compound from Salvia miltiorrhiza, has attracted significant attention for its therapeutic properties, including antitumor and antioxidant activities. However, its large-scale application is limited by the conventional production methods, which rely primarily on physical extraction and chemical synthesis. In this study, we constructed a de novo biosynthetic pathway for SAA by integrating two key modules: the HPPA hydroxylation module HpaBC (4-hydroxyphenylacetate 3-monooxygenase from Escherichia coli) and the DHPPA reductase module RgMDH (D-mandelic acid dehydrogenase from Rhodotorula graminis) into a high-yield tyrosine-producing strain, Tyr0, generating the engineered strain Dps01. Metabolic analysis identified the low catalytic efficiency of RgMDH as the critical bottleneck. To address this, structure-guided protein engineering of RgMDH was performed. Molecular dynamics (MD) simulations revealed that the optimal mutant RgMDH^M4 (T312A/F316S/T255I/A59H) exhibited a 10.3-fold increase in catalytic conformation occupancy compared to the wild-type (4.74% vs 0.46%), indicating enhanced substrate channel dynamics. This mutant exhibited a 295% increase in activity toward DHPPA, with enhanced substrate affinity and a 5.76-fold improvement in catalytic efficiency (k_cat/K_m). When integrated into strain Dps02, the engineered system achieved a maximum SAA production of 6.82 g/L within 34 hours, with a productivity of 0.21 g/L·h⁻¹ in a 5 L fed-batch fermentation. These results represent a significant advancement in the biosynthesis of SAA.