AbstractProtein engineering to expand the substrate spectrum of native enzymes opens new possibilities for bioproduction of valuable chemicals from non‐natural pathways. No natural microorganism can directly use sugars to produce 1,3‐propanediol (PDO). Here, we present a de novo route for the biosynthesis of PDO from sugar, which may overcome the mentioned limitations by expanding the homoserine synthesis pathway. The accomplishment of pathway from homoserine to PDO is achieved by protein engineering of glutamate dehydrogenase (GDH) and pyruvate decarboxylase to sequentially convert homoserine to 4‐hydroxy‐2‐ketobutyrate and 3‐hydroxypropionaldehyde. The latter is finally converted to PDO by using a native alcohol dehydrogenase. In this work, we report on experimental accomplishment of this non‐natural pathway, especially by protein engineering of GDH for the key step of converting homoserine to 4‐hydroxy‐2‐ketobutyrate. These results show the feasibility and significance of protein engineering for de novo pathway design and overproduction of desired industrial products.