Production of ( <i>R</i> )‐1,3‐butanediol via an Isozyme co‐expression Strategy in <i>Escherichia coli</i>

Microbial cell factories for the production of valuable chemicals often require multi-step biosynthetic pathways, whose efficiency is frequently limited by the performance and compatibility of pathway enzymes. Here, an isozyme co-expression strategy was proposed and evaluated in the (R)-1,3-BDO biosynthetic pathway of Escherichia coli. First, nine strains harboring different combinations of three candidate thiolases (AtoB, PhaA, Thl) and three candidate aldehyde dehydrogenases (Bld, Ald, AdhE2) were constructed and evaluated. Among them, the Thl-PhaB-Bld strain (B3-7) exhibited the highest (R)-1,3-BDO production (1.4 g/L). The remaining isozymes (AtoB, PhaA, Ald, and AdhE2) were then overexpressed individually in strain B3-7, with PhaA and AdhE2 showing the most significant improvement in (R)-1,3-BDO production. Co-expression of PhaA and AdhE2 further increased the (R)-1,3-BDO titer to 4.2 g/L, representing a 200% increase relative to strain B3-7. In addition, several metabolic effector molecules were screened as medium supplements, among which citric acid was identified as the most effective for improving (R)-1,3-BDO production. Finally, in glucose-feeding shake-flask fermentation, the isozyme co-expression strain produced 16.2 g/L (R)-1,3-BDO. Collectively, the isozyme co-expression strategy effectively enhanced (R)-1,3-BDO production and represents a promising approach for optimizing multi-step biosynthetic pathways.