Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering
In this study, to construct the riboflavin-producing strain R1, five key genes, ribA, ribB, ribC, ribD, and ribE, were cloned and ligated to generate the plasmid pET-AE, which was overexpressed in Escherichia coli BL21. The R1 strain accumulated 182.65 ± 9.04 mg/l riboflavin. Subsequently, the R2 st...
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| Format: | Article |
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Frontiers Media S.A.
2023-01-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1111790/full |
| _version_ | 1797952083802980352 |
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| author | Bing Fu Bing Fu Junhui Ying Qingwei Chen Qili Zhang Jiajie Lu Zhiwen Zhu Ping Yu |
| author_facet | Bing Fu Bing Fu Junhui Ying Qingwei Chen Qili Zhang Jiajie Lu Zhiwen Zhu Ping Yu |
| author_sort | Bing Fu |
| collection | DOAJ |
| description | In this study, to construct the riboflavin-producing strain R1, five key genes, ribA, ribB, ribC, ribD, and ribE, were cloned and ligated to generate the plasmid pET-AE, which was overexpressed in Escherichia coli BL21. The R1 strain accumulated 182.65 ± 9.04 mg/l riboflavin. Subsequently, the R2 strain was constructed by the overexpression of zwf harboring the constructed plasmid pAC-Z in the R1 strain. Thus, the level of riboflavin in the R2 strain increased to 319.01 ± 20.65 mg/l (74.66% increase). To further enhance ribB transcript levels and riboflavin production, the FMN riboswitch was deleted from E. coli BL21 with CRISPR/Cas9 to generate the R3 strain. The R4 strain was constructed by cotransforming pET-AE and pAC-Z into the R3 strain. Compared to those of E. coli BL21, the ribB transcript levels of R2 and R4 improved 2.78 and 3.05-fold, respectively. The R4 strain accumulated 437.58 ± 14.36 mg/l riboflavin, increasing by 37.17% compared to the R2 strain. These results suggest that the deletion of the FMN riboswitch can improve the transcript level of ribB and facilitate riboflavin production. A riboflavin titer of 611.22 ± 11.25 mg/l was achieved under the optimal fermentation conditions. Ultimately, 1574.60 ± 109.32 mg/l riboflavin was produced through fed-batch fermentation with 40 g/l glucose. This study contributes to the industrial production of riboflavin by the recombinant E. coli BL21. |
| first_indexed | 2024-04-10T22:41:56Z |
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| language | English |
| last_indexed | 2024-04-10T22:41:56Z |
| publishDate | 2023-01-01 |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-980c788ad90c4019b89656f0e4cd2e702023-01-16T05:00:01ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-01-011310.3389/fmicb.2022.11117901111790Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineeringBing Fu0Bing Fu1Junhui Ying2Qingwei Chen3Qili Zhang4Jiajie Lu5Zhiwen Zhu6Ping Yu7College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang, ChinaCollege of Forestry Science and Technology, Lishui Vocational and Technical College, Lishui, Zhejiang, ChinaCollege of Forestry Science and Technology, Lishui Vocational and Technical College, Lishui, Zhejiang, ChinaCollege of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang, ChinaCollege of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang, ChinaCollege of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang, ChinaCollege of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang, ChinaCollege of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang, ChinaIn this study, to construct the riboflavin-producing strain R1, five key genes, ribA, ribB, ribC, ribD, and ribE, were cloned and ligated to generate the plasmid pET-AE, which was overexpressed in Escherichia coli BL21. The R1 strain accumulated 182.65 ± 9.04 mg/l riboflavin. Subsequently, the R2 strain was constructed by the overexpression of zwf harboring the constructed plasmid pAC-Z in the R1 strain. Thus, the level of riboflavin in the R2 strain increased to 319.01 ± 20.65 mg/l (74.66% increase). To further enhance ribB transcript levels and riboflavin production, the FMN riboswitch was deleted from E. coli BL21 with CRISPR/Cas9 to generate the R3 strain. The R4 strain was constructed by cotransforming pET-AE and pAC-Z into the R3 strain. Compared to those of E. coli BL21, the ribB transcript levels of R2 and R4 improved 2.78 and 3.05-fold, respectively. The R4 strain accumulated 437.58 ± 14.36 mg/l riboflavin, increasing by 37.17% compared to the R2 strain. These results suggest that the deletion of the FMN riboswitch can improve the transcript level of ribB and facilitate riboflavin production. A riboflavin titer of 611.22 ± 11.25 mg/l was achieved under the optimal fermentation conditions. Ultimately, 1574.60 ± 109.32 mg/l riboflavin was produced through fed-batch fermentation with 40 g/l glucose. This study contributes to the industrial production of riboflavin by the recombinant E. coli BL21.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1111790/fullmetabolic engineeringriboflavinFMN riboswitchCRISPR/Cas9Escherichia coli BL21 |
| spellingShingle | Bing Fu Bing Fu Junhui Ying Qingwei Chen Qili Zhang Jiajie Lu Zhiwen Zhu Ping Yu Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering Frontiers in Microbiology metabolic engineering riboflavin FMN riboswitch CRISPR/Cas9 Escherichia coli BL21 |
| title | Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering |
| title_full | Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering |
| title_fullStr | Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering |
| title_full_unstemmed | Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering |
| title_short | Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering |
| title_sort | enhancing the biosynthesis of riboflavin in the recombinant escherichia coli bl21 strain by metabolic engineering |
| topic | metabolic engineering riboflavin FMN riboswitch CRISPR/Cas9 Escherichia coli BL21 |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1111790/full |
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