Sequential single-enzyme oxidation of 5-hydroxymethylfurfural to 2,5-furandicarboxylicacid by an engineered lanthanide-dependent alcohol dehydrogenase
2,5-furandicarboxylic acid (FDCA) is a potential platform chemical from renewable feedstocks to make various polymers and high-value compounds. Bioconversion of 5-hydroxymethylfurfural (HMF) to FDCA by 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 to FDCA, representing a rare and intriguing discovery. We then engineered the enzyme for improved thermostability and activity against HMF to improve FDCA yield. Using computational design method, the thermostability was first significantly improved, with the 4M variant obtained showing a Tm value increased by 9.6 oC, and a half-life improved by 10.3-fold compared to wild type. With the 4M as the template, potential mutation sites for engineering enzyme activity were identified based on calculation of 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 identified mutation targets. After five rounds evolution, a variant was obtained that produced FDCA at 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 are potentially used for high yield production of FDCA in industrial fields.
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