1,2,4-Butanetriol (BT) is an important precursor of energetic material 1,2,4-butanetriol trinitrate (BUN) in military field. Here, the recombinant Escherichia coil was modified by multi-strategy to increase BT production. First, the 2-keto acid reduction pathway was blocked by double deleting the genes yiaE and ycdW, resulting in 19% and 41% increase of BT titer and yield, respectively. Further disruption of xylose branch pathway by deleting xylA gene led to a 24% increase of BT yield, but the biomass and BT titer were decreased by 63% and 55%, respectively. To balance the carbon flux for cell growth and BT biosynthesis, the expression of xylA gene was interfered by antisense RNA. The biomass and BT titers of all the mutants harboring asRNA were restored and the highest BT production was improved by 214 %. Finally, substitute expression of the high efficient decarboxylase KivD led to a 72% increase of BT titer (10.03 g/L), which is significantly higher than previously reported (5.1 g/L). This work provided some effective strategies for improving BT production by balancing the carbon flux redistribution and reducing power regeneration. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.