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
Brown JE, Klement JF, McAllister WT (1986) Sequences of three promoters for the bacteriophage SP6 RNA polymerase. Nucl Acids Res 14:3521–3526
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
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
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
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
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
Papworth C, Bauer JC, Braman J, Wright DA (1996) QuikChange site-directed mutagenesis. Strategies 9:3–4
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
Shibui T, Uchida M, Teranishi Y (1988) A new hybrid promoter and its expression vector in E. coli. Agric Biol Chem 52:983–988
Sorensen HP (2010) Towards universal systems for recombinant gene expression. Microb Cell Fact 9:27
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
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
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
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.
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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