LAUSR

FDCA (2,5-furandicarboxylic acid) can be enzymatically converted from HMF (5-hydroxymethylfurfural). Pseudomonas putida S12 is promising for FDCA production, but generating stable P. putida S12 is difficult due to its polyploidy and lack of genome engineering tools. Here we showed that coupling CRISPR and λ-Red recombineering enabled one-step gene integration with high efficiency and frequency, and simultaneously replaced endogenous genes in all chromosomes. Using this approach, we generated two stable P. putida S12 strains expressing HMF/furfural oxidoreductase (HMFH) and HMF oxidase (HMFO), both being able to convert 50 mM HMF to ≈42-43 mM FDCA in 24 h. Co-supplementation of MnO2 and CaCO3 to the medium drastically improved the cell tolerance to HMF and enhanced FDCA production. Co-integrating HMFH and HMFT1 (HMF transporter) genes further improved FDCA production, enabling the cells to convert 250 mM HMF to 196 mM (30.6 g/L) FDCA in 24 h. This study implicates the potentials of CRISPR for generating stable P. putida S12 strains for FDCA production.