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

Abstract

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.

Strategies to increase the robustness of microbial cell factories

“…Another study found that the mutant RpoC H419P of the RNA polymerase subunit RpoC in an evolved E. coli was associated with its increased tolerance to octanoic acid (C8) and increased C8 production (Chen et al 2020). Using ALE coupled with genome restructuring technology, we obtained a robust astaxanthinproducing E. coli that was tolerant to large-scale culture high-density fermentation conditions (low pH, high osmolality (high NaCl concentration), high acetate concentration, and high reactive oxygen species concentration (high concentration of H 2 O 2 )), and found that rnb, envZ, and recC were associated with the robustness of the strain (Lu et al 2022).…”

Metabolic Engineering of Model Microorganisms for the Production of Xanthophyll

“…Based on these results, they knocked out the oxidative-stress-related genes ( uspE and yggE ) to elevate reactive oxygen species (ROS) in E. coli cells, thereby stimulating the production of astaxanthin to cope with oxidative stress [ 113 ]. Similarly, the knockout of some other pressure response proteins can also improve the production of astaxanthin to varying degrees [ 141 ]. Through transcriptome analysis, Wang et al [ 25 ] found that the mevalonate and ergosterol biosynthetic pathway, oxidative PPP, and antioxidant system were significantly down-regulated in the engineering of Y. lipolytica with astaxanthin overproduction.…”

Advances in the Discovery and Engineering of Gene Targets for Carotenoid Biosynthesis in Recombinant Strains

“…Adaptive laboratory evolution (ALE) proved to be a valuable approach to enhance the phenotype or physiological attributes of strains [34]. Indeed, researchers successfully identified numerous gene targets by using ALE; for example, the overexpression of the class E protein gene Did2 led to a 2.1-fold increase in β-carotene yields [37][38][39][40]. By employing ALE, our group also identified two novel gene targets, cho2 and pfk1, whose regulation can increase lycopene yield by 3.4 and 5.1 times, respectively [41,42].…”