Engineering an Mn(II)-oxidizing Pseudomonas whole-cell catalyst chassis to efficiently biosynthesize 2,5-furandicarboxylic acid from hydroxymethylfurfural.

2,5-Furandicarboxylic acid (FDCA) is a high-value chemical extensively used in the production of bio-based polymers, but bioconversion of furan derivatives like 5-hydroxymethylfurfural (HMF) into FDCA remains challenging owing to substrate cytotoxicity. Here, we engineered an Mn(II)-oxidizing Pseudomonas sp. MB04B for efficient FDCA biosynthesis from HMF. We deleted 4.6 % of the MB04B genome to generate the engineered MB04C-6 chassis, then introduced two exogenous gene cassettes, P_MP00-hmfH and P_J23119-hmfH'. Using the resulting MB04C-6/pHMF as a whole-cell catalyst, optimizing the reaction system, and incorporating CaCO₃ increased the FDCA yield by approximately 63.4-fold compared to MB04C-6. We also enhanced the CRISPR-associated transposases system for single-step chromosomal integration of exogenous genes. The optimal chassis strain MB04S-HMF8, rapidly produced 97 mmol/L FDCA from 100 mmol/L HMF in 12 h, with an FDCA production rate of 1.26 g L^-1h^-1, showcasing its potential as a robust, cost-effective, and environmentally sustainable whole-cell biocatalyst for industrial-scale FDCA production.