Metabolic Engineering of Escherichia Coli for Efficient Production of 1,4-Butanediol from Crude Glycerol

1,4-butanediol (1,4-BDO) is a significant non-natural chemical with diverse applications in various fields, particularly in the production of biodegradable plastic products as an essential monomer. The conversion of starch-based sugars using microbial cell factories provides a green alternative for the production of 1,4-BDO, but was limited by the low efficiency and high production cost. In this work, the classic CoA-dependent 1,4-BDO synthesis pathway was exogenously introduced into Escherichia coli, but the titer of 1,4-BDO was only 0.72g/L using glucose as the substrate. The poorly expressed enzymes and the ineffective distribution of central carbon flux were considered the main limiting factors. Combined genetic manipulation of the synthesis pathway and bioprocess engineering, glycerol was proven to be a more favorable feedstock for 1,4-BDO synthesis, and a novel pathway functioned by carboxylic acid reductase could achieve one-step catalysis from 4-hydroxybutyric acid to 4-hydroxybutyraldehyde, which avoided the rate-limiting steps involved by hydroxybutyrate CoA transferase (Cat2) and alcohol dehydrogenase (Bld). The titer of 1,4-BDO reached 1.31g/L and 1.07g/L in the novel pathway using pure glycerol and the industrial by-product crude glycerol as the substrate, respectively. The fed-batch fermentation was finally conducted, and the titer of 1,4-BDO was increased to 5.75g/L with a yield of 143.75mg/g glycerol. Our work removed the bottleneck of the 1,4-BDO synthesis pathway by introducing carboxylic acid reductase and effectively converted crude glycerol to 1,4-BDO, which enabled us to build a sustainable microbial cell factory to cost-efficiently produce 1,4-BDO as well as other biochemicals from crude glycerol.