A Multi-Functional Heterogeneous Biocatalyst for the Oxygen-Free Oxidative Condensation of Primary Alcohols into β-Hydroxy Acids

Enantiomerically pure β-hydroxy acids are valuable building blocks in polymer and fine chemical industries. However, biosynthetic routes to these compounds are limited by narrow feedstock availability. Here, we report a confined, cell-free biosynthetic pathway that converts primary alcohols into β-hydroxy acids using a multifunctional heterogeneous biocatalyst. Five enzymes were coimmobilized and spatially organized on glyoxyl-functionalized porous supports: an alcohol dehydrogenase from <i>Bacillus stearothermophilus</i>, a truncated CoA-acylating aldehyde dehydrogenase from <i>Salmonella enterica</i>, a thiolase from <i>Ralstonia eutropha</i>, a (<i>S</i>)-3-hydroxybutyryl-CoA dehydrogenase from <i>Thermus thermophilus</i> HB27, and a thioesterase from <i>Escherichia coli</i>. The system efficiently transforms ethanol into (<i>S</i>)-3-hydroxybutyric acid, achieving 18 mM of product, a 7-fold yield increase over soluble enzymes (2.54 mM) through a design-build-test-learn (DBTL) approach. Enzyme confinement promotes optimal cofactor gradients, supporting redox balance and driving this thermodynamically unfavorable cascade. The biocatalyst exhibits broad substrate scope, converting esters, aldehydes, and diols (i.e., ethylene glycol, potentially derived from plastic waste) into β-hydroxy acids. Operated in a packed-bed flow reactor, it retained over 50% productivity after 3 weeks. This work advances in vitro biocatalytic cascades using immobilized enzymes to convert simple feedstocks into high-value chiral molecules.