Upgrading Pseudomonas sp. toward Tolerance to a Synthetic BiomassHydrolysate Enrichedwith Furfural and 5‑Hydroxymethylfurfural

Several Pseudomonas species, including Pseudomonas putida KT2440, have a broad metabolic repertoire to assimilate biomass monomers such as lignin-derived compounds but struggle to tolerate biomass hydrolysates. Here, we examined the furan derivatives tolerance in a novel and nonpathogenic Pseudomonas species (strain BJa5) and in P. putida KT2440 using tolerance adaptive laboratory evolution (TALE) to enhance growth performance in a synthetic straw sugar cane hydrolysate enriched with furfural and 5-hydroxymethylfurfural (5-HMF). Initially, wild-type strains showed prolonged lag phases and low tolerance in the synthetic hydrolysate, but tolerance was improved after 90 days of sequential batch growth. Post-TALE, BJa5 and KT2440 end strains grew in synthetic hydrolysate containing 2 g/L furfural and 1 g/L 5-HMF at 48 and 24 h, respectively. Moreover, the KT2440 end strain notably grew in 2 g/L furfural and ≥1.7 g/L 5-HMF. Genome sequencing of end strains revealed mutations in genes and intergenic regions associated with transcriptional factors, acetate metabolism enzymes, environmental response proteins, and transposases. In a proof-of-concept experiment, the BJa5 end strain demonstrated the potential to detoxify synthetic hydrolysate by reducing the titers of acetate and furfural. This ability could enable industrial microorganisms, which are typically nontolerant to toxic hydrolysates, to be used for producing value-added compounds from biodetoxified hydrolysates.