Self-Sufficient In Vitro Multi-Enzyme Cascade for Efficient Synthesis of Danshensu froml-DOPA

Danshensu (DSS), a traditional Chinese medicine, is widely used for the treatment of cardiovascular and cancer diseases. Here, a one-pot multi-enzyme cascade pathway was designed for DSS synthesis from l-DOPA using tyrosine aminotransferase from Escherichia coli (EcTyrB) and d-isomer-specific 2-hydroxyacid dehydrogenase from Lactobacillus frumenti (LfD2-HDH). Glutamate dehydrogenase from Clostridium difficile (CdgluD) was also introduced for a self-sufficient system of α-ketoglutaric acid and NADH. Under optimal conditions (35 °C, pH 7.0, EcTyrB:LfD2-HDH:CdgluD = 3:2:1, glutamate:NAD⁺ = 1:1), 98.3% yield (at 20 mM l-DOPA) and space-time yield of 6.61 g L^-1 h^-1 (at 40 mM l-DOPA) were achieved. Decreased yields of DSS at elevated l-DOPA concentrations (100 mM) could be attributed to an inhibited CdgluD activity caused by NH₄⁺ accumulation. This developed multi-enzyme cascade pathway (including EcTyrB, LfD2-HDH, and CdgluD) provides an efficient and sustainable approach for the production of DSS from l-DOPA.