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ReToAd: simple method for the rapid replacement of promoters to improve protein production

Biotechnology Letters volume 40, pages 957–964 (2018)Cite this article

Abstract

Objective

To develop a method for fast replacement of promoters to improve protein production.

Results

A method (entitled retreat to advance or “ReToAd”), which includes a deleting PCR and a touchdown PCR, was validated by replacing seven IPTG-inducible promoters with enhanced green fluorescent protein (eGFP). The seven promoters were fully recovered by sequencing only 30 clones. The activity of E. coli harboring ω-transaminase (ω-TA) was increased from 112 U/mg cells (T7 promoter) to 147 U/mg cells (Trc promoter) by combining ReToAd and screening experiments. After screening a library comprising glutamate dehydrogenase (GDH) expressed by different promoters, the activity of E. coli cell harboring Trc-promoter-expressed GDH was ~31-fold higher than that of T7-promoter-expressed GDH.

Conclusions

The “ReToAd” for in situ rapid replacement of promoters was developed and optimized, and one round of “ReToAd” can be completed within 3 days.

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References

  • Arvani S, Markert A, Loeschcke A, Jaeger KE, Drepper T (2012) A T7 RNA polymerase-based toolkit for the concerted expression of clustered genes. J Biotechnol 159:162–171

    Article  PubMed  CAS  Google Scholar 

  • Brown JE, Klement JF, McAllister WT (1986) Sequences of three promoters for the bacteriophage SP6 RNA polymerase. Nucl Acids Res 14:3521–3526

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Cheng F, Zhu L, Lue H, Bernhagen J, Schwaneberg U (2014) Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas. Appl Microbiol Biotechnol 99:1237–1247

    Article  PubMed  CAS  Google Scholar 

  • Cheng F, Xu JM, Xiang C, Liu ZQ, Zhao LQ, Zheng YG (2017) Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis. Biotechnol Lett 39:567–575

    Article  PubMed  CAS  Google Scholar 

  • Cheng F, Tang X, Kardashliev T (2018) Transcription factor-based biosensors in high-throughput screening: advances and applications. Biotechnol J. https://doi.org/10.1002/biot.201700648

    Article  PubMed  Google Scholar 

  • Correa A, Oppezzo P (2011) Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening. Biotechnol J 6:715–730

    Article  PubMed  CAS  Google Scholar 

  • Liu ZQ, Dong LZ, Cheng F, Xue YP, Wang YS, Ding JN, Zheng YG, Shen YC (2011) Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10. J Agric Food Chem 59:11560–11570

    Article  PubMed  CAS  Google Scholar 

  • Papworth C, Bauer JC, Braman J, Wright DA (1996) QuikChange site-directed mutagenesis. Strategies 9:3–4

    Article  Google Scholar 

  • Savile CK, Janey JM, Mundorff EC, Moore JC, Tam S, Jarvis WR, Colbeck JC, Krebber A, Fleitz FJ, Brands J, Devine PN, Huisman GW, Hughes GJ (2010) Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329:305–309

    Article  PubMed  CAS  Google Scholar 

  • Shibui T, Uchida M, Teranishi Y (1988) A new hybrid promoter and its expression vector in E. coli. Agric Biol Chem 52:983–988

    CAS  Google Scholar 

  • Sorensen HP (2010) Towards universal systems for recombinant gene expression. Microb Cell Fact 9:27

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Vethanayagam JGG, Flower AM (2005) Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase. Microb Cell Fact 4:1–7

    Article  CAS  Google Scholar 

  • Yang JH, Ruff AJ, Hamer SN, Cheng F, Schwaneberg U (2016) Screening through the PLICable promoter toolbox enhances protein production in Escherichia coli. Biotechnol J 11:1639–1647

    Article  PubMed  CAS  Google Scholar 

  • Zhou J, Yang Y, Wang XM, Yu FB, Yu CL, Chen J, Cheng Y, Yan CQ, Chen JP (2013) Enhanced transgene expression in rice following selection controlled by weak promoters. BMC Biotechnol 13:29

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

This work was financially supported by Natural Science Foundation of China (31700693), Zhejiang Provincial Natural Science Foundation of China (LQ17C050002) and China Postdoctoral Science Foundation (2017M612030).

Supplementary information

Supplementary Table 1—Oligonucleotide sequences used for in removing and replacing promoters.

Supplementary Table 2—Oligonucleotide sequences used for sequencing.

Supplementary Figure 1—Maps of starting plasmids.

Supplementary Figure 2—ω-TA expression levels by different promoters.

Additional method 1—Expression levels of eGFP, ω-TA, NIT and GDH in MTPs or flasks.

Additional method 2—Protein analysis.

Additional method 3—Activity analysis of ω-transaminase, nitrilase, and glutamate dehydrogenase.

Author information

Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People’s Republic of China

    Feng Cheng, Chao Xiang, Xiao-Jian Zhang, Zhi-Qiang Liu & Yu-Guo Zheng

Authors
  1. Feng Cheng
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  2. Chao Xiang
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  3. Xiao-Jian Zhang
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  4. Zhi-Qiang Liu
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  5. Yu-Guo Zheng
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Corresponding author

Correspondence to Zhi-Qiang Liu.

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The authors declare no financial or commercial conflict of interest.

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Cheng, F., Xiang, C., Zhang, XJ. et al. ReToAd: simple method for the rapid replacement of promoters to improve protein production. Biotechnol Lett 40, 957–964 (2018). https://doi.org/10.1007/s10529-018-2541-4

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  • DOI: https://doi.org/10.1007/s10529-018-2541-4

Keywords

  • IPTG-inducible promoters
  • Protein production
  • Replacing promoters
  • ReToAd