Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli
PDF

Keywords

Amylases
Carbohydrate debranching enzyme
Enzyme activity
Expression vector
Gene expression
in starch hydrolysis
industrial applications
Pullulanase fermentation
SD sequence
Semi-quantitative RT-PCR
Stem-loop structure

How to Cite

1.
Li T, Ding Y, Zhang J, Jiao G, Sun L, Liu Z, Qiu L. Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli. Electron. J. Biotechnol. [Internet]. 2017 Oct. 17 [cited 2024 Oct. 13];29(1). Available from: https://www.ejbiotechnology.info/index.php/ejbiotechnology/article/view/2017.07.001

Abstract

Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5′ or 3′ untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression.

Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5′ SD sequence at the 5′ UTR of pulA. pulA-3t was constructed by the addition of a 3′ stem-loop structure at the 3′ UTR of pulA. SD-pulA-3t was constructed by the addition of the 5′ SD sequence at the 5′ UTR and a 3′ stem-loop structure at the 3′ UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively.

Conclusion: The addition of the 5′ SD sequence at the 5′ UTR and a 3′ stem-loop structure at the 3′ UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.

PDF

Upon acceptance of an article by the journal, authors will be asked to transfer the copyright to Electronic Journal of Biotechnology, which is committed to maintain the electronic access to the journal and to administer a policy of fair control and ensure the widest possible dissemination of the information. The author can use the article for academic purposes, stating clearly the following: "Published in Electronic Journal of Biotechnology at DOI:10.2225/volXX-issueX-fulltext-XX".

The Copyright Transfer Agreement must be submitted as a signed scanned copy to biotec@ucv.cl. All authors must send a copy of this document.