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

Biotechnology for Biofuels

Linjiang Zhu¹  Yin Li²  Zhen Cai² 

[1] Key Laboratory of Industrial Biotechnology, Ministry of Education of China, School of Biotechnology, Jiangnan University, Wuxi 214122, China

[2] 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

关键词: n-butanol;    Stress tolerance;    Synthetic evolution module;    Genetic toggle switch;    Stress-induced mutagenesis;    Adaptive evolution;   

Others  :  1219148
DOI  :  10.1186/s13068-015-0276-1

 received in 2014-09-17, accepted in 2015-06-18,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Zhu et al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

【 参考文献 】

• [1]Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, et al.: Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 2006, 440:940-3.
• [2]Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, et al.: Programming cells by multiplex genome engineering and accelerated evolution. Nature 2009, 460:894-U133.
• [3]Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, et al.: Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 2009, 27:753-U107.
• [4]Khalil AS, Collins JJ: Synthetic biology: applications come of age. Nat Rev Genet 2010, 11:367-79.
• [5]Zhang Y, Zhu Y, Li Y: The importance of engineering physiological functionality into microbes. Trends Biotechnol 2009, 27:664-72.
• [6]Zhu L, Zhu Y, Zhang Y, Li Y: Engineering the robustness of industrial microbes through synthetic biology. Trends Microbiol 2012, 20:94-101.
• [7]Kitano H: Biological robustness. Nat Rev Genet 2004, 5:826-37.
• [8]Rosche WA, Foster PL: The role of transient hypermutators in adaptive mutation in Escherichia coli. Proc Natl Acad Sci U S A 1999, 96:6862-7.
• [9]Foster PL: Stress-induced mutagenesis in bacteria. Crit Rev Biochem Mol Biol 2007, 42:373-97.
• [10]Galhardo RS, Hastings PJ, Rosenberg SM: Mutation as a stress response and the regulation of evolvability. Crit Rev Biochem Mol Biol 2007, 42:399-435.
• [11]Gibson JL, Lombardo MJ, Thornton PC, Hu KH, Galhardo RS, Beadle B, et al.: The sigma(E) stress response is required for stress-induced mutation and amplification in Escherichia coli. Mol Microbiol 2010, 77:415-30.
• [12]Cohen SE, Walker GC: The transcription elongation factor NusA is required for stress-induced mutagenesis in Escherichia coli. Curr Biol 2010, 20:80-5.
• [13]Al Mamun AM, Lombardo MJ, Shee C, Lisewski AM, Gonzalez C, Lin DX, et al.: Identity and function of a large gene network underlying mutagenic repair of DNA breaks. Science 2012, 338:1344-8.
• [14]Zhu L, Cai Z, Zhang Y, Li Y. Engineering stress tolerance of Escherichia coli by stress-induced-mutagenesis (SIM) based adaptive evolution. Biotechnol J. 2014;9.
• [15]Gardner TS, Cantor CR, Collins JJ: Construction of a genetic toggle switch in Escherichia coli. Nature 2000, 403:339-42.
• [16]Shetty RP, Endy D, Knight TF Jr: Engineering BioBrick vectors from BioBrick parts. J Biol Eng 2008, 2:5. BioMed Central Full Text
• [17]Cairns J, Foster PL: Adaptive reversion of a frameshift mutation in Escherichia coli. Genetics 1991, 128:695-701.
• [18]Jin C, Yao MF, Liu HF, Lee CFF, Ji J: Progress in the production and application of n-butanol as a biofuel. Renew Sust Energ Rev 2011, 15:4080-106.
• [19]Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, Liao JC: Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl Environ Microbiol 2011, 77:2905-15.
• [20]Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R: Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemicals. Nature 2011, 476:355-9.
• [21]Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, et al.: Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 2008, 10:305-11.
• [22]Luan GD, Cai Z, Li Y, Ma YH: Genome replication engineering assisted continuous evolution (GREACE) to improve microbial tolerance for biofuels production. Biotechnol Biofuels 2013, 6:137. BioMed Central Full Text
• [23]Elowitz MB, Leibler S: A synthetic oscillatory network of transcriptional regulators. Nature 2000, 403:335-8.
• [24]You LC, Cox RS, Weiss R, Arnold FH: Programmed population control by cell-cell communication and regulated killing. Nature 2004, 428:868-71.
• [25]Kobayashi H, Kaern M, Araki M, Chung K, Gardner TS, Cantor CR, et al.: Programmable cells: interfacing natural and engineered gene networks. Proc Natl Acad Sci U S A 2004, 101:8414-9.
• [26]Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, et al.: Engineering Escherichia coli to see light—these smart bacteria ‘photograph’ a light pattern as a high-definition chemical image. Nature 2005, 438:441-2.
• [27]Cagatay T, Turcotte M, Elowitz MB, Garcia-Ojalvo J, Suel GM: Architecture-dependent noise discriminates functionally analogous differentiation circuits. Cell 2009, 139:512-22.
• [28]Shapiro JA: Observations on the formation of clones containing araB-lacZ cistron fusions. Mol Gen Genet 1984, 194:79-90.
• [29]Goho S, Bell G: Mild environmental stress elicits mutations affecting fitness in Chlamydomonas. Proc Biol Sci 2000, 267:123-9.
• [30]Badyaev AV: Stress-induced variation in evolution: from behavioural plasticity to genetic assimilation. Proc Biol Sci 2005, 272:877-86.
• [31]Rosenberg SM, Shee C, Frisch RL, Hastings PJ: Stress-induced mutation via DNA breaks in Escherichia coli: a molecular mechanism with implications for evolution and medicine. Bioessays 2012, 34:885-92.
• [32]Shee C, Gibson JL, Darrow MC, Gonzalez C, Rosenberg SM: Impact of a stress-inducible switch to mutagenic repair of DNA breaks on mutation in Escherichia coli. Proc Natl Acad Sci U S A 2011, 108:13659-64.
• [33]LeClerc JE, Li B, Payne WL, Cebula TA: High mutation frequencies among Escherichia coli and Salmonella pathogens. Science 1996, 274:1208-11.
• [34]Giraud A, Matic I, Tenaillon O, Clara A, Radman M, Fons M, et al.: Costs and benefits of high mutation rates: adaptive evolution of bacteria in the mouse gut. Science 2001, 291:2606-8.
• [35]Denamur E, Lecointre G, Darlu P, Tenaillon O, Acquaviva C, Sayada C, et al.: Evolutionary implications of the frequent horizontal transfer of mismatch repair genes. Cell 2000, 103:711-21.
• [36]Fuchs RP, Fujii S, Wagner J: Properties and functions of Escherichia coli: Pol IV and Pol V. Adv Protein Chem 2004, 69:229-64.
• [37]Hastings PJ, Hersh MN, Thornton PC, Fonville NC, Slack A, Frisch RL, et al.: Competition of Escherichia coli DNA polymerases I, II and III with DNA Pol IV in stressed cells. PLoS One 2010., 5Article ID e10862
• [38]Cox MM: Regulation of bacterial RecA protein function. Crit Rev Biochem Mol Biol 2007, 42:41-63.
• [39]Foster PL: Adaptive mutation in Escherichia coli. Cold Spring Harb Symp Quant Biol 2000, 65:21-9.
• [40]McKenzie GJ, Lee PL, Lombardo MJ, Hastings PJ, Rosenberg SM: SOS mutator DNA polymerase IV functions in adaptive mutation and not adaptive amplification. Mol Cell 2001, 7:571-9.
• [41]Layton JC, Foster PL: Error-prone DNA polymerase IV is controlled by the stress-response sigma factor, RpoS, in Escherichia coli. Mol Microbiol 2003, 50:549-61.
• [42]Lombardo MJ, Aponyi I, Rosenberg SM: General stress response regulator RpoS in adaptive mutation and amplification in Escherichia coli. Genetics 2004, 166:669-80.