Fully biocatalytic stereodivergent conversion of levulinic acid to enantiopure γ-valerolactone

γ-Valerolactone (GVL) is a biomass-derived platform chemical with broad industrial applications, while its enantiopure forms are valuable chiral building blocks for fine-chemical synthesis. Here, we developed a fully biocatalytic Escherichia coli whole-cell cascade for the stereodivergent conversion of levulinic acid (LA) to enantiopure ( R )- or ( S )-GVL. The modular system integrates stereocomplementary LA reduction by engineered dehydrogenases, formate dehydrogenase (FDH)-dependent nicotinamide cofactor regeneration, and 4-hydroxyvalerate (4HV) lactonization by periplasmically targeted engineered paraoxonase-1 (ePON1). Periplasmic targeting enhanced apparent whole-cell GVL formation from 4HV by 8.2-fold relative to the non-targeted construct. The integrated one-pot whole-cell cascades produced ( R )-GVL and ( S )-GVL in 64.5% and 52.0% yields, respectively, with >99.9% enantiomeric excess for both products, exceeding the maximum 4HV-to-GVL lactonization fraction previously reported for a PON1-based biological GVL production system. The cascade was further applied to LA-containing streams generated from food-waste-derived biomass. These streams naturally contained co-produced formate at approximately 1.0–1.5 molar equivalents relative to LA, closely matching the stoichiometric reducing-equivalent requirement for FDH-dependent cofactor regeneration and enabling bioconversion without exogenous formate supplementation. Using these biomass-derived streams, GVL yields reached up to 38.3% and 70.0% through the ( R )- and ( S )-routes, respectively. Importantly, formate, which would otherwise contribute to the downstream separation burden during LA purification, was directly utilized as an intrinsic reducing-power source, potentially reducing both separation requirements and the need for external formate supplementation. These results establish a modular whole-cell strategy for highly enantioselective and stereodivergent GVL production while coupling renewable LA upgrading with direct valorization of a biomass-derived coproduct.