Sequential regulation of dual catalytic modules for the directed biosynthesis of 2,5-furandicarboxylic acid in Escherichia coli

Bioconversion of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA) confers advantages of mild conditions, high selectivity, and eco-friendliness. However, enzymatic catalysis is expensive and multi-strain microbial catalysis is limited by the transmembrane transport of intermediates between strains, restricting the efficient synthesis of FDCA and generating a byproduct. In this study, we aimed to establish sequential regulation of dual catalytic modules in Escherichia coli to precisely control the expression of hydroxymethylfurfural oxidase (HMFO) and vanillin dehydrogenase (VDH1), enabling the directed one-pot conversion of HMF to FDCA with no detectable accumulation of the measured intermediates and simplifying the operational process. At a 15 mM HMF scale, this strategy achieved a FDCA yield of 100%, significantly higher than the 78.6% yield of the dual-enzyme cascade system established by HMFO and VDH1, and the 56.73% yield from the dual-strain catalysis constructed by two engineered E. coli strains that harbor either hmfo or vdh1(Student's t-test, p < 0.0001). Furthermore, at a laboratory scale (production of a fermentation broth containing 100 g of FDCA), life cycle assessment demonstrated sequential regulation of dual catalytic modules represented the lowest environmental impact among the three systems (dual-strain catalysis and dual-enzyme cascade). It reduced global warming potential, fossil fuel depletion, and ecotoxicity by 70-82% compared with the other two systems. Overall, this study establishes a more efficient and sustainable strategy for directed FDCA biosynthesis.