Efficient Biosynthesis of Ectoine in Recombinant Escherichia coli by Biobrick Method
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Plasmids
2.2. Construction of the Expression Plasmids
2.3. Heterogeneous Protein Purification
2.4. Protein Expression Analysis
2.5. Ectoine Detection
2.6. Optimizing Conditions for Ectoine Biosynthesis
2.6.1. Optimization of pH
2.6.2. Reaction Temperature
2.6.3. L-aspartic Acid Concentration
2.6.4. Optimization of KCl
2.6.5. Mass-to-Volume Ratio of Cell Pellets
2.6.6. Reaction Time
2.6.7. Protein Induction
2.6.8. Effect of Promoter
2.6.9. Bacterial Reusability
2.7. Stress Resistance Effects
2.7.1. Salt Tolerance
2.7.2. pH Tolerance
2.8. Sequencing Analysis
3. Results
3.1. Construction of Co-Expression Vectors
3.2. Purification Analysis of EctA, EctB, and EctC Recombinant Proteins
3.3. Optimizing Conditions for First Screening with T7 Promoter
3.4. Effect of Promoter
3.4.1. Optimization Conditions for Second Screening under Different Promoters
3.4.2. Comparison of Ectoine Production among Different Recombinant Strains
3.5. Protein Expression Analysis
3.6. Saline Alkali Tolerance Effects
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primers. | Target Sequence (5′→3′) | Description |
---|---|---|
EctA F01 EctA R01 EctB-F01 EctB-R01 EctC-F01 EctC-R01 | GCATATGTGGGAATTAGTTAATC CCTCGAGCCTTAATGGTCCAATTC GCATATGAA ACA AACTGA TATG AGAGCTCGTTAGCAACAGGCTCAG GCATATGAA AGT AGTAGCTCT ACTCGAGTTCGTCATCAACTACTG | ectA gene cloning ectB gene cloning ectC gene cloning |
Strain Type | Ectoine Amount (mg/mL/d) | Disadvantages or Features | Approach/Method | Reference |
---|---|---|---|---|
H. elongate DSM142 | 7.4 | salinity issues, low yield | bacterial milking | [14] |
H. salina BCRC17875 | 7.61 | salinity issues, low yield | bacterial milking | [15] |
E. coli BW25113 | 25.1 | salinity issues, low yield | bacterial milking | [24] |
E. coli MG1655 | 12.7 | complicated, low yield | whole cell catalysis | [25] |
E. coli W3110 | 31.37 | complicated operational design | fed-batch process | [26] |
H. salina DSM 5928T | 14.86 | low yield, complicated design | fed-batch process | [30] |
E. coli BL21(DE3) | 167.2 | high yield, efficient design | biobrick approach | This study |
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Naeem, M.; Yuan, H.; Luo, S.; Zhang, S.; Wei, X.; He, G.; Zhao, B.; Ju, J. Efficient Biosynthesis of Ectoine in Recombinant Escherichia coli by Biobrick Method. Fermentation 2024, 10, 450. https://doi.org/10.3390/fermentation10090450
Naeem M, Yuan H, Luo S, Zhang S, Wei X, He G, Zhao B, Ju J. Efficient Biosynthesis of Ectoine in Recombinant Escherichia coli by Biobrick Method. Fermentation. 2024; 10(9):450. https://doi.org/10.3390/fermentation10090450
Chicago/Turabian StyleNaeem, Muhammad, Huiling Yuan, Suya Luo, Simei Zhang, Xinyue Wei, Guangzheng He, Baohua Zhao, and Jiansong Ju. 2024. "Efficient Biosynthesis of Ectoine in Recombinant Escherichia coli by Biobrick Method" Fermentation 10, no. 9: 450. https://doi.org/10.3390/fermentation10090450