Self-Sustaining Cell-Free Artificial Biocatalytic Cascades for the Synthesis of Chiral β-Hydroxy Acids from Simple Feedstocks

The increasing demand for sustainable synthesis of chiral building blocks has driven the development of cell-free biocatalytic cascades capable of transforming simple feedstocks into high-value molecules. Here, we present the design of self-sustaining in vitro biosynthetic systems for the production of enantiopure β-hydroxy acids through CoA-dependent pathways integrating cofactor recycling and enzyme confinement strategies. Enantiomerically pure β-hydroxy acids are valuable building blocks in polymer and fine chemical industries.

As starting materials, two strategies were attempted, firstly, vinyl esters were exploited as dual acyl and electron donors in an abiotic thioesterification and a four-enzyme cascade involving non-decarboxylative Claisen condensation, reduction, hydrolysis, and in situ NADH recycling. By embedding both activation energy and reducing power within the substrate, the system efficiently produced (S)-3-hydroxybutyrate without external ATP supply while simultaneously recycling CoASH and NADH. Second strategy consisted in the oxygen-free oxidative condensation of primary alcohols trough a multifunctional heterogeneous biocatalyst developed by the coimmobilization and spatial organization of five enzymes on glyoxyl-functionalized porous supports. The enzyme confinement promoted favorable cofactor gradients and enhanced the thermodynamic feasibility of the cascade.

Together, these studies highlight the potential of engineered in vitro biocatalytic cascades and immobilized enzyme systems to achieve efficient, atom-economical, and scalable synthesis of valuable chiral molecules from simple and sustainable feedstocks.