Improving Furfural Tolerance of Escherichia coli by Integrating Adaptive Laboratory Evolution with CRISPR-Enabled Trackable Genome Engineering (CREATE)

Genetic diversity is an important factor affecting the efficiency of adaptive laboratory evolution (ALE). The recent development of precise tools and strategies for genomic engineering has greatly accelerated mutant library construction for ALE. Here, a global regulator library based on the CRISPR-enabled trackable genome engineering (CREATE) technology was first used to accelerate adaptive evolution for improved furfural tolerance in Escherichia coli, and the furfural tolerance was increased approximately 2-fold in the genetically diversified CREATE-based strains. The evolved strain tolerated up to 4.7 g/L furfural and also showed marked cross-tolerance to NaCl, isobutanol, butanol, ethanol, and high temperature. Whole-genome sequencing and mutant reconstruction analysis revealed for the first time that rpoB(P153L) mutation leads to greatly increased furfural tolerance. The expression of genes coding central carbon and energy metabolism was significantly altered according to transcriptome analysis. In particular, it was confirmed for the first time that the knockout of sRNA sgrS and the overexpression of sRNA arrS significantly increased furfural tolerance. This study provides evidence that combined ALE and the CREATE technology can not only obtain highly efficient strains with favorable mutation combinations but also accelerate ALE by providing much greater genomic and functional diversity.