Table 4.

Representative examples of mutant library construction for ALE.

	Strategy and description	Characteristics of evolved strains	References
Genetic diversification by introducing non-targeted mutations	Traditional physical and chemical mutagenesis by adding chemical reagents or by X-ray, γ-ray, ultraviolet (UV), Atmospheric and Room Temperature Plasma (ARTP) and Atmospheric Pressure Glow Discharge (APGD) treatment	The combination of the adenine analog 2-aminopurine (2AP) and cytidine analog zebularine (ZEB) increased the mutation frequency of the rpoB gene 35-fold to 53,000×108.	(Ang et al. 2016)
	Increased mutation rate and DNA exchange by recursive protoplast fusion (genome shuffling) with multiple parental strains for combinatorial phenotypes.	After three rounds of genome recombination and 5 months of ALE, evolved strains X2 and X5 produced 20 and 50% higher ethanol concentrations than th starting strain at 40°C, respectively.	(Lin et al. 2019)
	Increase the global mutation rate by overexpressing mutated genes in the genome or mutated genes on mutagenic plasmid vectors (including MP6, TaGTEAM, MutaT7, EvolvR, PACE, Retrons, etc.) such as Genome Replication Engineering Assisted Continuous Evolution (GREACE)	The use of eMutaT7 increased the minimum inhibitory concentration of the antibiotic cefotaxime for the strai from 0.06 to 800 mg/L. The minimum inhibitory concentration for ceftazidime was increased from 0.2 mg/L to 4 g/L.	(Park & Kim 2021)
	Increasing global mutation rates by disruption of the native DNA mismatch repair system, introducing mutant DNA polymerases or heterogenous R/M system.	A mutant library was constructed by deleting the mutS gene encoding the DNA mismatch repair enzyme in L. casei, and an evolved strain that could grow well at pH 2.5 with a 10-fold higher survival rate was obtained.	(Overbeck et al. 2017)
Genetic diversification by introducing in vitro generated targeted DNA variants	Global Transcriptional Mechanism Engineering (gTME) utilizes an error-prone plasmid library of global cellular transcriptional machinery to induce global perturbations of the transcriptome and unlock complex phenotypes.	A mutant library of the rpoA was constructed in E. coli by gTME, and a total of 409 mutants were obtained. The mutant strain BTR1:BRM-353 was able to produce 104.3 mg/L of indigo, representing a 204% increase.	(Alper & Stephanopoulos 2007)
	Obtain mutated DNA from customized synthetic oligo pool, when used as integration donor fragments, multiplex automated genome engineering (MAGE) and CRISPR-enabled trackable genome engineering (CREATE) are created and applied for genome-wide mapping of mutations at single or multi-nucleotide resolution.	The mutant Crp S28P, which was able to tolerate 2 g/L furfural, was obtained by combining CREATE with ALE. The biomass of the evolved strain increased by 50% compared with the wild type after 8 h of growth.	(Garst et al. 2017)