Sequential single-enzyme oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylic acid by an engineered lanthanide-dependent alcohol dehydrogenase

Abstract

2,5-Furandicarboxylic acid (FDCA) is a potential platform chemical available from renewable feedstocks to make various polymers and high-value compounds. Bioconversion of 5-hydroxymethylfurfural (HMF) to FDCA with biocatalysts has the advantages of high selectivity, cost-effectiveness, and eco-friendliness. However, the full oxidation of HMF often requires a combination of two or three enzymes, as the production of FDCA involves three consecutive oxidation steps. In this study, we identified a pyrroloquinoline quinone (PQQ) and lanthanide-dependent alcohol dehydrogenase, PedH, capable of directly converting HMF into FDCA, representing a rare and intriguing discovery. We then engineered the enzyme for improved thermostability and activity against HMF to improve the FDCA yield. Using a computational design method, the thermostability was first significantly improved, with the 4M variant obtained showing a T_m value increased by 9.6 °C, and a half-life improved by 10.3-fold compared to the wild type. With 4M as the template, potential mutation sites for engineering enzyme activity were identified based on calculations of the binding free energy and a deep learning model, MutCompute. An automatic high-throughput platform for site-saturation mutagenesis library construction and screening was developed and applied to the mutation targets identified. After five rounds of evolution, a variant was obtained that produced FDCA in a yield 474-fold higher than that of 4M. Under optimized reaction conditions, the optimal variant achieved a FDCA yield of 96.4% with 40 mM HMF as the substrate. Molecular dynamics simulations revealed that the mutations expanded the substrate binding pocket and shortened the reaction distances between substrate and cofactors. This study provides a highly efficient evolution approach for PedH, and several variants that can potentially be used for the high-yield production of FDCA in industrial applications.