Engineering a homoserine-derived non-natural pathway allows heterologous production of 1,3-propanediol (1,3-PDO) from glucose without adding expensive vitamin B. Due to the lack of efficient enzymes to catalyze the deamination of homoserine and the decarboxylation of 4-hydroxy-2-ketobutyrate, the previously engineered strain can only produce 51.5 mg/L 1,3-PDO using homoserine and glucose as cosubstrates. In this study, we systematically screened the enzymes from different protein families to catalyze the two corresponding reactions and further optimized the selected enzymes by protein engineering. Together with the improvement of homoserine supply by systematic metabolic engineering, an engineered Escherichia coli strain with an optimal combination of aspartate transaminase ( aspC) from E. coli, pyruvate decarboxylase ( pdc) from Zymomonas mobiliz, and alcohol dehydrogenase yqhD from E. coli can produce 0.32 g/L 1,3-PDO from glucose in shake flask cultivation. The titer of 1,3-PDO was further increased to 0.49 g/L or 0.63 g/L by introducing a point mutation of I472A into pdc gene or constructing a fusion protein between aspC and pdc. This study lays the basis for developing a potential process for 1,3-PDO production from sugars without using expensive coenzyme B.