Self-sufficient Biocatalytic Cascade for the Continuous Synthesis of Danshensu in Flow

A new strategy has been developed to successfully produce the active component danshensu ex vivo. For this purpose, phenylalanine dehydrogenase from Bacillus sphaericus was combined with the novel hydroxyphenylpyruvate reductase from Mentha x piperita, thereby providing an in situ cofactor regeneration throughout the conversion process. The purified enzymes were co-immobilized and subsequently employed in batch biotransformation, resulting in 60% conversion of 10 mM L-dopa within 24 h, with a catalytic amount of NAD as cofactor. Furthermore, the bienzymatic system was implemented as a packed-bed reactor in continuous flow, achieving a conversion rate up to 80% with 60 min retention time. The process was further intensified by implementing a 48-h flow bioreaction. The biocatalysts demonstrated remarkable stability, retaining 62% of their initial activity at the end of the process. The final productivity of the isolated compound (96% purity) was calculated to be 1.84 g L h yielding a sustainable synthesis of danshensu. KEY POINTS: • Characterization of the hydroxyphenylpyruvate reductase from Mentha x piperita • Bi-enzymatic system in a cascade reaction to produce danshensu • Purification and isolation of the active compound danshensu.