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 rpoBP153L 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.