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Background Astaxanthin is one of the strongest antioxidants in nature and has been widely used in aquaculture, food, cosmetic and pharmaceutical industries. Numerous stresses caused in the process of a large scale-culture, such as high acetate concentration, high osmolarity, high level of reactive oxygen species, high glucose concentration and acid environment, etc., limit cell growth to reach the real high cell density, thereby affecting astaxanthin production. Results We developed an adaptive laboratory evolution (ALE) strategy to enhance the production of chemicals by improving strain tolerance against industrial fermentation conditions. This ALE strategy resulted in 18.5% and 53.7% increases in cell growth and astaxanthin production in fed-batch fermentation, respectively. Whole-genome resequencing showed that 65 mutations with amino acid substitution were identified in 61 genes of the shuffled strain  Escherichia coli  AST-4AS. CRISPR interference (CRISPRi) and activation (CRISPRa) revealed that the shuffled strain with higher astaxanthin production may be associated with the mutations of some stress response protein genes, some fatty acid biosynthetic genes and  rppH . Repression of  yadC, ygfI  and  rcsC , activation of  rnb, envZ  and  recC  further improved the production of astaxanthin in the shuffled strain  E. coli  AST-4AS. Simultaneous deletion of  yadC  and overexpression of  rnb  increased the production of astaxanthin by 32% in the shuffled strain  E. coli  AST-4AS. Conclusion This ALE strategy will be powerful in engineering microorganisms for the high-level production of chemicals.

Adaptive laboratory evolution and shuffling of Escherichia coli to enhance its tolerance and production of astaxanthin