High specificity of MsmCAR toward 4-hydroxyvaleric acid enables efficient 1,4-pentanediol production from...

Alkanediols are industrial compounds with diverse applications. However, the biological production of C5 diols is limited by the low substrate specificity and efficiency of carboxylic acid reductases (CARs) toward short to medium-chain substrates. We developed a whole-cell biotransformation system for efficient 1,4-pentanediol (1,4-PDO) production, aimed at developing a platform pathway for diol biosynthesis. By screening 10 CARs, we identified MsmCAR from Mycolicibacterium smegmatis MC2 155 as the most effective for C5 diol biosynthesis, with high specificity toward 4-hydroxyvaleric acid (4-HV). We constructed a whole-cell catalyst expressing MsmCAR and enhanced its performance by introducing ppk2b from Corynebacterium glutamicum ATCC 13032 and chnD from Acinetobacter sp., which encode proteins involved in cofactor regeneration. Systematic optimization of the reaction conditions including buffers, cofactors, metal ions, and cultivation parameters led to a maximum titer of 78.10 mM 1,4-PDO, with a productivity of 1.86 mM/h, when using 4-HV as a substrate. To expand the substrate scope to levulinic acid, 3-hydroxybutyrate dehydrogenase and formate dehydrogenase biosynthetic genes were introduced. This system yielded 22.10 mM 1,4-PDO within 10 h. This work highlights the potential of MsmCAR as a versatile biocatalyst and provides a sustainable strategy for producing short- to medium-chain diols from biomass-derived feedstocks.