Adaptive laboratory evolution of Lactiplantibacillus plantarum enhances furan tolerance and lactic acid production in lignocellulosic hydrolysate

Furfural and 5-hydroxymethylfurfural (HMF) are two major lignocellulosic growth inhibitors that hinder microbial growth and fermentation of lignocellulosic hydrolysate for lactic acid production. In this study, we employed adaptive laboratory evolution (ALE) to enhance the tolerance of Lactiplantibacillus plantarum JGR2, a strain previously isolated in our lab, to furfural and HMF. The adapted strains demonstrated significantly improved growth in the presence of these inhibitors compared to the parental strains. Whole-genome resequencing revealed multiple mutations including high-impact non-conservative mutations in genes encoding DNA recombination and repair protein (RecF, lp_0005), flavin prenyltransferase (UbiX lp_0271), and oligo-1,6-glucosidase (lp_0189). Transcriptomic analysis indicated that adaptation elicited more pronounced differential gene expression compared to acute inhibitor exposure. Upon furfural exposure, the furfural-adapted isolate showed fewer differentially expressed genes than the parental strain, indicating a possible shift in the transcriptomic profile as a possible mechanism of furfural adaptation. Mechanistic investigation revealed that the adapted isolates reduce furfural into the less toxic furfuryl alcohol, suggesting a key detoxification mechanism. Notably, lp_3051 (dhaT, 1,3-propanediol dehydrogenase) encoding furfural reductase activity was upregulated in both furfural- and HMF-adapted isolates. Membrane fatty acid analysis revealed increased unsaturated fatty acids and cyclopropane fatty acids in adapted strains. Finally, the adapted strains exhibited improved growth in rice straw hydrolysate and produced significantly higher relative lactic acid yields compared to the parental strain, thus demonstrating improved bioproduction under inhibitor-rich conditions. This study not only provides a comprehensive understanding of L. plantarum's response to lignocellulosic inhibitors, but also yields evolved bacterial candidates for further scientific and industrial exploration.