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Background During fermentation, industrial microorganisms encounter multiple stresses that inhibit cell growth and decrease fermentation yields, in particular acid stress, which is due to the accumulation of acidic metabolites in the fermentation medium. Although the addition of a base to the medium can counteract the effect of acid accumulation, the engineering of acid-tolerant strains is considered a more intelligent and cost-effective solution. While synthetic biology theoretically provides a novel approach for devising such tolerance modules, in practice it is difficult to assemble stress-tolerance modules from hundreds of stress-related genes. Results In this study, we designed a set of synthetic acid-tolerance modules for fine-tuning the expression of multi-component gene blocks comprising a member of the proton-consuming acid resistance system ( gadE ), a periplasmic chaperone ( hdeB ), and reactive oxygen species (ROS) scavengers ( sodB  and  katE ). Directed evolution was used to construct an acid-responsive  asr  promoter library, from which four variants were selected and used in the synthetic modules. The module variants were screened in a stepwise manner under mild acidic conditions (pH 5–6), first by cell growth using the laboratory  Escherichia coli  strain MG1655 cultured in microplates, and then by lysine production performance using the industrial lysine-producing  E. coli  strain MG1655 SCEcL3 cultured first in multiple 10-mL micro-bioreactors, and then in 1.3-L parallel bioreactors. The procedure resulted in the identification of a best strain with lysine titer and yield at pH 6.0 comparable to the parent strain at pH 6.8. Conclusion Our results demonstrate a promising synthetic-biology strategy to enhance the growth robustness and productivity of  E. coli  upon the mildly acidic conditions, in both a general lab strain MG1655 and an industrial lysine-producing strain SCEcL3, by using the stress-responsive synthetic acid-tolerance modules comprising a limited number of genes. This study provides a reliable and efficient method for achieving synthetic modules of interest, particularly in improving the robustness and productivity of industrial strains.

Synthetic acid stress-tolerance modules improve growth robustness and lysine productivity of industrial Escherichia coli in fermentation at low pH