Figure 2

Flowchart of the Genome Replication Engineering Assisted Continuous Evolution (GREACE) method. Proofreading element (PE) of the genome replication machinery was selected to generate a mutant library (PEM-lib). For phenotype-improvement, PEM-lib was transformed into host cells, and cultivated in conditions with gradually increased selective strengths (media in flasks with colours from light blue to dark blue). Evolved strains with the most beneficial and adaptive mutation accumulations (red sparks represented for beneficial mutations and black sparks for detrimental mutations) would show the best adaption advantages and dominate the PEM-lib populations. Genetically modified PE mutant would be eliminated from the evolved strains to stabilize the obtained genotypes and phenotypes.