Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system
Abstract Background Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is sti...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-05-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-020-01370-9 |
| _version_ | 1828462179719839744 |
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| author | Manman Sun Xiong Gao Zihao Zhao An Li Yali Wang Yankun Yang Xiuxia Liu Zhonghu Bai |
| author_facet | Manman Sun Xiong Gao Zihao Zhao An Li Yali Wang Yankun Yang Xiuxia Liu Zhonghu Bai |
| author_sort | Manman Sun |
| collection | DOAJ |
| description | Abstract Background Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. Results In this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L. Conclusion In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield. |
| first_indexed | 2024-12-11T02:31:46Z |
| format | Article |
| id | doaj.art-1c9bd3b4ae4142e6866d25bee982293d |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-11T02:31:46Z |
| publishDate | 2020-05-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-1c9bd3b4ae4142e6866d25bee982293d2022-12-22T01:23:49ZengBMCMicrobial Cell Factories1475-28592020-05-0119111210.1186/s12934-020-01370-9Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression systemManman Sun0Xiong Gao1Zihao Zhao2An Li3Yali Wang4Yankun Yang5Xiuxia Liu6Zhonghu Bai7National Engineering Laboratory of Cereal Fermentation Technology, Jiangnan UniversityDivision of Life Science and Center for Chinese Medicine, Hong Kong University of Science and TechnologyNational Engineering Laboratory of Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory of Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory of Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory of Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory of Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory of Cereal Fermentation Technology, Jiangnan UniversityAbstract Background Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. Results In this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L. Conclusion In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.http://link.springer.com/article/10.1186/s12934-020-01370-9Corynebacterium glutamicumRecombinant proteinBCD expression systemScreening fore-cistron sequencesPΙNP |
| spellingShingle | Manman Sun Xiong Gao Zihao Zhao An Li Yali Wang Yankun Yang Xiuxia Liu Zhonghu Bai Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system Microbial Cell Factories Corynebacterium glutamicum Recombinant protein BCD expression system Screening fore-cistron sequences PΙNP |
| title | Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system |
| title_full | Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system |
| title_fullStr | Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system |
| title_full_unstemmed | Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system |
| title_short | Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system |
| title_sort | enhanced production of recombinant proteins in corynebacterium glutamicum by constructing a bicistronic gene expression system |
| topic | Corynebacterium glutamicum Recombinant protein BCD expression system Screening fore-cistron sequences PΙNP |
| url | http://link.springer.com/article/10.1186/s12934-020-01370-9 |
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