Limosilactobacillus reuteri produces 3-hydroxypropionaldehyde (3-HPA), which has broad-spectrum antimicrobial activity and potential as a natural food preservative. However, 3-HPA is cytotoxic, and this limits their production of 3-HPA. We used adaptive laboratory evolution to develop a novel strain, L. reuteri NK45, by selection of cells with enhanced tolerance to 3-HPA. These cells maintained high viability during bioconversion in the presence of ascorbic acid, enabling sustained 3-HPA production. When conducted in 800 mM glycerol, 100 mM ascorbic acid, and 1 mM cyanocobalamin at 30°C an0d an OD600 100 for 2 h, the reaction yielded a record titer 592.0 ± 3.8 mM 3-HPA. The produced 3-HPA had strong antimicrobial activity against foodborne and pathogenic microorganisms, with minimum inhibitory concentrations of 1 to 3 mM. These findings demonstrate that L. reuteri NK45 has efficient and sustainable biological production of food-grade 3-HPA, and highlight its potential as an industrial strain for natural preservative production.