Abstract
The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5′ untranslated region (5′-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5′-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6–14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48–817-fold) translation initiation rate. No effect of the 5′-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5′-UTR. Interestingly, the amount of this transcript was 2.6–3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5′-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5′-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5′-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.
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Acknowledgments
This work was supported by the MIT-Portugal Program, Fundação para a Ciência e a Tecnologia (grant UID/BIO/04565/2013 awarded to iBB—Institute for Bioengineering and Biosciences and PhD grant SFRH/BD/33786/2009 to Michaela Šimčíková).
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Šimčíková, M., Alves, C.P.A., Brito, L. et al. Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase. Appl Microbiol Biotechnol 100, 6725–6737 (2016). https://doi.org/10.1007/s00253-016-7565-x
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DOI: https://doi.org/10.1007/s00253-016-7565-x