Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production
Abstract Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wi...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2021-09-01
|
| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-021-01680-6 |
| _version_ | 1818735892905328640 |
|---|---|
| author | Fei Du Yun-Qi Liu Ying-Shuang Xu Zi-Jia Li Yu-Zhou Wang Zi-Xu Zhang Xiao-Man Sun |
| author_facet | Fei Du Yun-Qi Liu Ying-Shuang Xu Zi-Jia Li Yu-Zhou Wang Zi-Xu Zhang Xiao-Man Sun |
| author_sort | Fei Du |
| collection | DOAJ |
| description | Abstract Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins. |
| first_indexed | 2024-12-18T00:28:29Z |
| format | Article |
| id | doaj.art-bc4de17ab7a34d69aa41d26fd3d23997 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-18T00:28:29Z |
| publishDate | 2021-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-bc4de17ab7a34d69aa41d26fd3d239972022-12-21T21:27:11ZengBMCMicrobial Cell Factories1475-28592021-09-0120111010.1186/s12934-021-01680-6Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein productionFei Du0Yun-Qi Liu1Ying-Shuang Xu2Zi-Jia Li3Yu-Zhou Wang4Zi-Xu Zhang5Xiao-Man Sun6School of Food Science and Pharmaceutical Engineering, Nanjing Normal UniversityNanjing Foreign Language SchoolSchool of Food Science and Pharmaceutical Engineering, Nanjing Normal UniversitySchool of Food Science and Pharmaceutical Engineering, Nanjing Normal UniversitySchool of Food Science and Pharmaceutical Engineering, Nanjing Normal UniversitySchool of Food Science and Pharmaceutical Engineering, Nanjing Normal UniversitySchool of Food Science and Pharmaceutical Engineering, Nanjing Normal UniversityAbstract Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.https://doi.org/10.1186/s12934-021-01680-6BL21 (DE3)Inducible promoterT7 RNA polymeraseRecombinant protein |
| spellingShingle | Fei Du Yun-Qi Liu Ying-Shuang Xu Zi-Jia Li Yu-Zhou Wang Zi-Xu Zhang Xiao-Man Sun Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production Microbial Cell Factories BL21 (DE3) Inducible promoter T7 RNA polymerase Recombinant protein |
| title | Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production |
| title_full | Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production |
| title_fullStr | Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production |
| title_full_unstemmed | Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production |
| title_short | Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production |
| title_sort | regulating the t7 rna polymerase expression in e coli bl21 de3 to provide more host options for recombinant protein production |
| topic | BL21 (DE3) Inducible promoter T7 RNA polymerase Recombinant protein |
| url | https://doi.org/10.1186/s12934-021-01680-6 |
| work_keys_str_mv | AT feidu regulatingthet7rnapolymeraseexpressioninecolibl21de3toprovidemorehostoptionsforrecombinantproteinproduction AT yunqiliu regulatingthet7rnapolymeraseexpressioninecolibl21de3toprovidemorehostoptionsforrecombinantproteinproduction AT yingshuangxu regulatingthet7rnapolymeraseexpressioninecolibl21de3toprovidemorehostoptionsforrecombinantproteinproduction AT zijiali regulatingthet7rnapolymeraseexpressioninecolibl21de3toprovidemorehostoptionsforrecombinantproteinproduction AT yuzhouwang regulatingthet7rnapolymeraseexpressioninecolibl21de3toprovidemorehostoptionsforrecombinantproteinproduction AT zixuzhang regulatingthet7rnapolymeraseexpressioninecolibl21de3toprovidemorehostoptionsforrecombinantproteinproduction AT xiaomansun regulatingthet7rnapolymeraseexpressioninecolibl21de3toprovidemorehostoptionsforrecombinantproteinproduction |