Objectives 3,4-Dihydroxybutyric acid (3,4-DHBA) is a multifunctional C4 platform compound widely used for the synthesis of various materials, including pharmaceuticals. Although, a biosynthetic pathway for 3,4-DHBA production has been developed, its low yield still precludes large-scale use. Here, a heterologous four-step biosynthetic pathway was established in recombinant Escherichia coli (E. coli) using a combinatorial strategy. Results Several aldehyde dehydrogenases (ALDHs) were screened, using in vitro enzyme assays, to identify suitable catalysts for the dehydrogenation of 3,4-dihydroxybutanal (3,4-DHB) to 3,4-DHBA. A pathway containing glucose dehydrogenase (BsGDH) from Bacillus subtilis, d-xylonate dehydratase (YagF) from E. coli, benzoylformate decarboxylase (PpMdlC) from Pseudomonas putida and ALDH was introduced into E. coli, generating 3.04 g/L 3,4-DHBA from d-xylose (0.190 g 3,4-DHBA/g d-xylose). Disruption of competing pathways by deleting xylA, ghrA, ghrB and adhP contributed to an 87% increase in 3,4-DHBA accumulation. Expression of a fusion construct containing PpMdlC and YagF enhanced the 3,4-DHBA titer, producing the highest titer and yield reported thus far (7.71 g/L; 0.482 g 3,4-DHBA/g d-xylose). Conclusions These results showed that deleting genes from competing pathways and constructing fusion proteins significantly improved the titer and yield of 3,4-DHBA in engineered E. coli.