2,5-Furandicarboxylic acid (FDCA) is a versatile chemical for polymer synthesis, involving poly(ethylene 2,5-furandicarboxylate) (PEF), a promising alternative for poly(ethylene terephthalate) (PET). Recently, FDCA can be chemically obtained from biobased 5-hydroxymethyl furfural (HMF), whose process requires an environmental-unfriendly condition such as high temperature/pressure, organic solvents and metal-based catalysts. Alternatively, Pseudomonas putida (P. putida) S12 is promising for FDCA production, but engineering P. putida remains challenging, probably due to the lack of genetic engineering tool. In this study, we developed a smart coupling of Lambda-Red recombinase and CRISPR-Cas nuclease to generate stable engineered P. putida S12 for FDCA production. We first evaluated 3 Cas orthologs and revealed their efficient ability of inducing double-stranded breaks (DSBs), resulting in a robust cell death of 99%. We further discovered that P. putida S12 lacked DNA repair machinery, leading to the impossibility of foreign gene integration within the cell genome, even with the assistance of DSBs. Subsequent development of Lambda-Red-coupled CRISPR could facilitate the gene integration into all endogenous copies of target gene with extremely high efficiency and frequency and thus, resulting in the stable cell line, which can robustly maintain the integrated gene in serial selection-free subcultures. This strategy was then utilized to integrate HMF/furfural oxidoreductase (HMFH) or HMF oxidase (HMFO) into the genome of P. putida. Resultantly, both engineered strains were able to produce ~42-43 mM FDCA from 50 mM HMF. Moreover, we uncovered the cosupplement of CaCO₃ and MnO₂ drastically enhanced the cell tolerance to HMF and thus, increase FDCA production to ~177 mM within 24 hours, corresponding to the titer of ~26 g/L and the productivity of 1.08 g/L.h. Together, the data imply the potential of CRISPR technology for gene engineering in P. putida S12 for FDCA production.

Yu-Chen Hu

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