Enhanced cascade biosynthesis of D-phenyllactic acid using metal-organic framework-encapsulated co-expressing E. coli.

Phenylpyruvic acid, as a versatile organic acid, has attracted widespread attention in the fields of food, feed, pharmaceuticals, and cosmetics for its synthetic methods. In this study, we co-expressed L-amino acid oxidase, D-lactate dehydrogenase, and glucose dehydrogenase in Escherichia coli. The resulting recombinant whole-cell biocatalysts exhibited high efficiency in the cascade enzymatic synthesis of D-phenyllactic acid from L-phenylalanine. However, their limitations in operational stability and reusability have impeded their broader application. To address this issue, the co-expressing bacteria was immobilized on the metal-organic framework (MOF) ZIF-90. The encapsulation rate of the immobilized E. coli cells (E. coli@ZIF-90) and the recovery rate of their catalytic activity were 94.8 % and 92.7 %, respectively. The physical and biochemical properties of the E. coli@ZIF-90 were subsequently studied in detail. Compared with free cells, E. coli@ZIF-90 demonstrates superior stability under acidic and alkaline conditions, enhanced thermal stability, and increased tolerance to metal ions and organic reagents. After storage at 4°C for 8 days, the residual enzyme activity of the immobilized cells is still over 75 %, which is about 1.7 times that of free cells. Following 10 cycles of biocatalytic reactions, the immobilized cells maintained over 80 % of their enzyme activity, contrasting with the 58.6 % residual activity observed in free cells. In the enzyme cascade reaction catalyzed by E. coli@ZIF-90, 25 g·l L-PHE was completely reacted to produce D-PLA 17.8 g·l, with an enantiomeric excess (e.e.) greater than 99.5 %. This study provides a promising method for obtaining efficient and robust biocatalysts for the biosynthesis of D-PLA. Additionally, it provides essential insights that are extendable to the synthesis of a broader range of compounds.