title: Development of a stress-induced mutagenesis module for autonomous adaptive evolution of Escherichia coli to improve its stress tolerance.
abstract: BACKGROUND
  Microbial tolerance to different environmental stresses is of importance for efficient production of biofuels and biochemical. Such traits are often improved by evolutionary engineering approaches including mutagen-induced mutagenesis and successive passage. In contrast to these approaches which generate mutations in rapidly growing cells, recent research showed that mutations could be generated in non-dividing cells under stressful but non-lethal conditions, leading to the birth of the theory of stress-induced mutagenesis (SIM). A molecular mechanism of SIM has been elucidated to be mutagenic repair of DNA breaks. This inspired us to develop a synthetic SIM module to simulate the mutagenic cellular response so as to accelerate microbial adaptive evolution for an improved stress tolerance.
  
  
  RESULTS
  A controllable SIM evolution module was devised based on a genetic toggle switch in Escherichia coli. The synthetic module enables expression and repression of the genes related to up- and down-regulation responses during SIM in a bistable way. Upon addition of different inducers, the module can be turned on or off, triggering transition to a mutagenic or a high-fidelity state and thus allowing periodic adaptive evolution. Six genes (recA, dinB, umuD, ropS, ropE, and nusA) in the up-regulation responses were evaluated for their potentials to enhance the SIM rate. Expression of recA, dinB, or ropS alone increased the SIM rate by 4.5- to 13.7-fold, whereas their combined expression improved the rate by 31.9-fold. Besides, deletion of mutL increased the SIM rate by 82-fold. Assembly of these genes into the SIM module in the mutL-deletion E. coli strain elevated the SIM rate by nearly 3000-fold. Accelerated adaptive evolution of E. coli equipped with this synthetic SIM module was demonstrated under n-butanol stress, with the minimal inhibitory concentration of n-butanol increasing by 56 % within 2.5 months.
  
  
  CONCLUSIONS
  A synthetic SIM module was constructed to simulate cellular mutagenic responses during SIM. Based on this, a novel evolutionary engineering approach-SIM-based adaptive evolution-was developed to improve the n-butanol tolerance of E. coli.
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101 China ; Key Laboratory of Industrial Biotechnology, Ministry of Education of China, School of Biotechnology, Jiangnan University, Wuxi, 214122 China.
authors.forename: Linjiang
authors.initials: L
authors.lastname: Zhu
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101 China.
authors.forename: Yin
authors.initials: Y
authors.lastname: Li
authors.affiliation: CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101 China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cai
keywords: Adaptive evolution; Genetic toggle switch; Stress tolerance; Stress-induced mutagenesis; Synthetic evolution module; n-butanol