Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli
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...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2017-09-01
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| Series: | Electronic Journal of Biotechnology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0717345817300416 |
| _version_ | 1818358676957691904 |
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| author | Tao Li Yatong Ding Jun Zhang Guobao Jiao Lipeng Sun Zhongmin Liu Liyou Qiu |
| author_facet | Tao Li Yatong Ding Jun Zhang Guobao Jiao Lipeng Sun Zhongmin Liu Liyou Qiu |
| author_sort | Tao Li |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-13T20:32:48Z |
| format | Article |
| id | doaj.art-226e59a88f6e469585619113da86f73e |
| institution | Directory Open Access Journal |
| issn | 0717-3458 |
| language | English |
| last_indexed | 2024-12-13T20:32:48Z |
| publishDate | 2017-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Electronic Journal of Biotechnology |
| spelling | doaj.art-226e59a88f6e469585619113da86f73e2022-12-21T23:32:23ZengElsevierElectronic Journal of Biotechnology0717-34582017-09-0129C636710.1016/j.ejbt.2017.07.001Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coliTao Li0Yatong Ding1Jun Zhang2Guobao Jiao3Lipeng Sun4Zhongmin Liu5Liyou Qiu6College of Life Sciences, Henan Agricultural University, Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Zhengzhou 450002, ChinaCollege of Life Sciences, Henan Agricultural University, Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Zhengzhou 450002, ChinaCollege of Life Sciences, Henan Agricultural University, Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Zhengzhou 450002, ChinaHenan Yangshao Biochemical Engineering Co., Ltd, Mianchi 472400, ChinaHenan Yangshao Biochemical Engineering Co., Ltd, Mianchi 472400, ChinaHenan Yangshao Biochemical Engineering Co., Ltd, Mianchi 472400, ChinaCollege of Life Sciences, Henan Agricultural University, Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture, Zhengzhou 450002, ChinaBackground: 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.http://www.sciencedirect.com/science/article/pii/S0717345817300416AmylasesCarbohydrate debranching enzymeEnzyme activityExpression vectorGene expressionStarch hydrolysisIndustrial applicationsPullulanase fermentationSD sequenceSemi-quantitative RT-PCRStem-loop structure |
| spellingShingle | Tao Li Yatong Ding Jun Zhang Guobao Jiao Lipeng Sun Zhongmin Liu Liyou Qiu Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli Electronic Journal of Biotechnology Amylases Carbohydrate debranching enzyme Enzyme activity Expression vector Gene expression Starch hydrolysis Industrial applications Pullulanase fermentation SD sequence Semi-quantitative RT-PCR Stem-loop structure |
| title | Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli |
| title_full | Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli |
| title_fullStr | Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli |
| title_full_unstemmed | Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli |
| title_short | Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli |
| title_sort | improving the expression of recombinant pullulanase by increasing mrna stability in escherichia coli |
| topic | Amylases Carbohydrate debranching enzyme Enzyme activity Expression vector Gene expression Starch hydrolysis Industrial applications Pullulanase fermentation SD sequence Semi-quantitative RT-PCR Stem-loop structure |
| url | http://www.sciencedirect.com/science/article/pii/S0717345817300416 |
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