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Development of a stress-induced mutagenesis module for autonomous adaptive evolution of Escherichia coli to improve its stress tolerance.

Zhu L, Li Y, Cai Z - Biotechnol Biofuels (2015)

Bottom Line: 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.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.Based on this, a novel evolutionary engineering approach-SIM-based adaptive evolution-was developed to improve the n-butanol tolerance of E. coli.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Improving n-butanol tolerance of E. coli by periodic adaptive evolution via the SIM module. a The scheme. b The n-butanol MIC after each cycle of evolution. The strain SMB07/pML/pTL16 equipped with the SIM module and the control strain FC40/pACYC184/pTAD without the module were compared. c-f Growth curves of the evolved strain SMB705/pML/pTL16 and its parent strain SMB07/pML/pTL16 at 37 °C in various media: c LB medium with various concentrations of n-butanol; d LB medium with 50 or 60 g/L NaCl; e LB medium at pH 4.8, adjusted by lactic acid; f M9 minimal medium containing 0.2 % glucose. For all the media, 100 μg/mL ampicillin, 25 μg/mL tetracycline, and 500 ng/mL aTc were added. All the OD600 values were detected using 200 μL of culture in 96-well microplates. Three independent cultures were prepared for each condition
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Fig4: Improving n-butanol tolerance of E. coli by periodic adaptive evolution via the SIM module. a The scheme. b The n-butanol MIC after each cycle of evolution. The strain SMB07/pML/pTL16 equipped with the SIM module and the control strain FC40/pACYC184/pTAD without the module were compared. c-f Growth curves of the evolved strain SMB705/pML/pTL16 and its parent strain SMB07/pML/pTL16 at 37 °C in various media: c LB medium with various concentrations of n-butanol; d LB medium with 50 or 60 g/L NaCl; e LB medium at pH 4.8, adjusted by lactic acid; f M9 minimal medium containing 0.2 % glucose. For all the media, 100 μg/mL ampicillin, 25 μg/mL tetracycline, and 500 ng/mL aTc were added. All the OD600 values were detected using 200 μL of culture in 96-well microplates. Three independent cultures were prepared for each condition

Mentions: n-Butanol is an important renewable biofuel that has attracted much attention [18]. Metabolic engineering has enabled E. coli to produce n-butanol at the level of grams per liter, which is competitive with the productivity of the industrial producer, Clostridium acetobutylicum [19–21]. However, the tolerance of E. coli to n-butanol limited further increase of the titer [22]. To address this issue, the strain SMB07/pML/pTL16 equipped with the SIM module was subjected to adaptive evolution under the stress of n-butanol (Fig. 4a).Fig. 4


Development of a stress-induced mutagenesis module for autonomous adaptive evolution of Escherichia coli to improve its stress tolerance.

Zhu L, Li Y, Cai Z - Biotechnol Biofuels (2015)

Improving n-butanol tolerance of E. coli by periodic adaptive evolution via the SIM module. a The scheme. b The n-butanol MIC after each cycle of evolution. The strain SMB07/pML/pTL16 equipped with the SIM module and the control strain FC40/pACYC184/pTAD without the module were compared. c-f Growth curves of the evolved strain SMB705/pML/pTL16 and its parent strain SMB07/pML/pTL16 at 37 °C in various media: c LB medium with various concentrations of n-butanol; d LB medium with 50 or 60 g/L NaCl; e LB medium at pH 4.8, adjusted by lactic acid; f M9 minimal medium containing 0.2 % glucose. For all the media, 100 μg/mL ampicillin, 25 μg/mL tetracycline, and 500 ng/mL aTc were added. All the OD600 values were detected using 200 μL of culture in 96-well microplates. Three independent cultures were prepared for each condition
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487801&req=5

Fig4: Improving n-butanol tolerance of E. coli by periodic adaptive evolution via the SIM module. a The scheme. b The n-butanol MIC after each cycle of evolution. The strain SMB07/pML/pTL16 equipped with the SIM module and the control strain FC40/pACYC184/pTAD without the module were compared. c-f Growth curves of the evolved strain SMB705/pML/pTL16 and its parent strain SMB07/pML/pTL16 at 37 °C in various media: c LB medium with various concentrations of n-butanol; d LB medium with 50 or 60 g/L NaCl; e LB medium at pH 4.8, adjusted by lactic acid; f M9 minimal medium containing 0.2 % glucose. For all the media, 100 μg/mL ampicillin, 25 μg/mL tetracycline, and 500 ng/mL aTc were added. All the OD600 values were detected using 200 μL of culture in 96-well microplates. Three independent cultures were prepared for each condition
Mentions: n-Butanol is an important renewable biofuel that has attracted much attention [18]. Metabolic engineering has enabled E. coli to produce n-butanol at the level of grams per liter, which is competitive with the productivity of the industrial producer, Clostridium acetobutylicum [19–21]. However, the tolerance of E. coli to n-butanol limited further increase of the titer [22]. To address this issue, the strain SMB07/pML/pTL16 equipped with the SIM module was subjected to adaptive evolution under the stress of n-butanol (Fig. 4a).Fig. 4

Bottom Line: 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.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.Based on this, a novel evolutionary engineering approach-SIM-based adaptive evolution-was developed to improve the n-butanol tolerance of E. coli.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus