Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification
Abstract Background 5-Aminolevulinic acid (ALA) recently received much attention due to its potential application in many fields. In this study, an ALA production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on known regulatory and metab...
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| Format: | Article |
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BMC
2022-10-01
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| Series: | Journal of Biological Engineering |
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| Online Access: | https://doi.org/10.1186/s13036-022-00307-7 |
| _version_ | 1798030008193646592 |
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| author | Changchuan Ye Yuting Yang Xi Chen Lijie Yang Xia Hua Mengjie Yang Xiangfang Zeng Shiyan Qiao |
| author_facet | Changchuan Ye Yuting Yang Xi Chen Lijie Yang Xia Hua Mengjie Yang Xiangfang Zeng Shiyan Qiao |
| author_sort | Changchuan Ye |
| collection | DOAJ |
| description | Abstract Background 5-Aminolevulinic acid (ALA) recently received much attention due to its potential application in many fields. In this study, an ALA production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on known regulatory and metabolic information and CRISPR/Cas9 mediated gene knockout. Results A metabolic strategy to produce ALA directly from glucose in this recombinant E. coli via the C5 pathway was applied herein. The rational metabolic engineering by gene knockouts significantly improved ALA production from 662.3 to 1601.7 mg/L. In addition, we managed to synergistically produce ALA via the C4 pathway in recombinant strain. The expression of a modified hemA gene, encoding an ALA synthase from Rhodobacter sphaeroides, improved ALA production from 1601.7 to 2099.7 mg/L. After 24 h cultivation, a yield of 0.210 g ALA per g glucose was achieved by constructed E. coli D5:FYABD-RSA. Conclusion Our study revealed that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification and optimization of gene expression. |
| first_indexed | 2024-04-11T19:34:29Z |
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| id | doaj.art-bd55f3d2d7ce455abbd23af8c0da8aae |
| institution | Directory Open Access Journal |
| issn | 1754-1611 |
| language | English |
| last_indexed | 2024-04-11T19:34:29Z |
| publishDate | 2022-10-01 |
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| spelling | doaj.art-bd55f3d2d7ce455abbd23af8c0da8aae2022-12-22T04:06:55ZengBMCJournal of Biological Engineering1754-16112022-10-0116111510.1186/s13036-022-00307-7Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modificationChangchuan Ye0Yuting Yang1Xi Chen2Lijie Yang3Xia Hua4Mengjie Yang5Xiangfang Zeng6Shiyan Qiao7State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural UniversityState Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural UniversityState Key Laboratory for Agro-Biotechnology, and Ministry of Agriculture and Rural Affairs, Key Laboratory for Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural UniversityState Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural UniversityState Key Laboratory for Agro-Biotechnology, and Ministry of Agriculture and Rural Affairs, Key Laboratory for Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural UniversityNational Feed Engineering Technology Research CentreState Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural UniversityState Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry Centre, China Agricultural UniversityAbstract Background 5-Aminolevulinic acid (ALA) recently received much attention due to its potential application in many fields. In this study, an ALA production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on known regulatory and metabolic information and CRISPR/Cas9 mediated gene knockout. Results A metabolic strategy to produce ALA directly from glucose in this recombinant E. coli via the C5 pathway was applied herein. The rational metabolic engineering by gene knockouts significantly improved ALA production from 662.3 to 1601.7 mg/L. In addition, we managed to synergistically produce ALA via the C4 pathway in recombinant strain. The expression of a modified hemA gene, encoding an ALA synthase from Rhodobacter sphaeroides, improved ALA production from 1601.7 to 2099.7 mg/L. After 24 h cultivation, a yield of 0.210 g ALA per g glucose was achieved by constructed E. coli D5:FYABD-RSA. Conclusion Our study revealed that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification and optimization of gene expression.https://doi.org/10.1186/s13036-022-00307-7E. coli5-Aminolevulinic AcidCRISPR/cas9T7 Expression SystemMetabolic engineering |
| spellingShingle | Changchuan Ye Yuting Yang Xi Chen Lijie Yang Xia Hua Mengjie Yang Xiangfang Zeng Shiyan Qiao Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification Journal of Biological Engineering E. coli 5-Aminolevulinic Acid CRISPR/cas9 T7 Expression System Metabolic engineering |
| title | Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification |
| title_full | Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification |
| title_fullStr | Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification |
| title_full_unstemmed | Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification |
| title_short | Metabolic engineering of Escherichia coli BW25113 for the production of 5-Aminolevulinic Acid based on CRISPR/Cas9 mediated gene knockout and metabolic pathway modification |
| title_sort | metabolic engineering of escherichia coli bw25113 for the production of 5 aminolevulinic acid based on crispr cas9 mediated gene knockout and metabolic pathway modification |
| topic | E. coli 5-Aminolevulinic Acid CRISPR/cas9 T7 Expression System Metabolic engineering |
| url | https://doi.org/10.1186/s13036-022-00307-7 |
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