Toward Cost-Effective and Sustainable Production of 2,5-Bis(hydroxymethyl)furan by an Engineered Alcohol...

2,5-Bis(hydroxymethyl)furan (BHMF) is a useful intermediate for manufacturing biobased polymers, biofuels, and fine chemicals. Biocatalytic reduction of 5-hydroxymethylfurfural (HMF) obtained via hexoses dehydration is a sustainable route toward BHMF, but the demand for efficient and cost-effective processes remains highly pressing. In this work, cofactor specificity engineering of an alcohol dehydrogenase from Escherichia coli (EcYjgB) was performed to improve its catalytic efficiency toward NADH, thus allowing the use of cheaper formate instead of glucose as the cosubstrate for BHMF production from HMF. Upon two-round mutagenesis, a promising variant V2 (S199D/S200R) was achieved, with greatly improved catalytic efficiencies toward both NADH and HMF as well as with reduced substrate inhibition. The recombinant E. coli strain harboring the variant and formate dehydrogenase displayed a much higher catalytic performance in HMF reduction at the expense of formate, compared to the strain incorporating the wild-type enzyme. The practicability of the variant V2 was demonstrated by preparative-scale synthesis of BHMF at 0.5-1 M substrate loadings with 7.8-9.8 g/L h productivities and >98% yields. According to environmental footprint evaluation and preliminary feedstock cost analysis, the formate-fueled bioprocess may hold great potential for cost-effective and sustainable production of BHMF.