Engineering aldehyde dehydrogenase PaALDH70140 from Pseudomonas aeruginosa PC-1 with improved catalytic properties for 5-hydroxyme-thyl-2-furancarboxylic acid synthesis

The catalytic transformation of biomass-derived 5-hydroxymethylfurfural (HMF) into value-added chemicals and biofuels has received considerable interest. Previously, we demonstrated that Pseudomonas aeruginosa PC-1 could be used for selective 900 mM HMF oxidation into 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) with over 90% yield. Here, we aimed to mine the potential aldehyde dehydrogenases (ALDHs) for HMF biocatalysis and improved the catalytic activity and tolerance through protein engineering. ALDHs were heterologously expressed in E. coli BL21 (DE3) and subsequently the most efficient HMF oxidation conditions of E. coli/pET-PaALDH70140 were identified. Based on the structural analysis of PaALDH70140 with the HMF and NAD+ complexes, the engineering hotspots in the active site architecture were identified and subjected to saturation mutagenesis. The mutant L172R displayed 175% higher HMF catalytic efficiency than PaALDH70140, and the mutant also showed improved HMF-tolerance. Kinetic parameters suggested that L172R had higher HMF affinity and catalytic efficiency, indicating a higher HMF conversion efficiency than the wild-type. Molecular docking revealed that the mutation introduced new hydrogen bonds between the enzyme and HMF, which fixed the spatial position of HMF and shortened the reaction time of the catalysis, thus enhancing the catalytic efficiency of HMF. In the fedbatch process, E. coli/pET-PaALDH70140-L172R exhibited a 166% increase in the yield of HMFCA within 22 h, proving its potential for HMF bioconversion applications.