Enhancing the Production of Pinene in Escherichia coli by Using a Combination of Shotgun, Product-Tolerance and I-SceI Cleavage Systems
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Strains, Plasmids and Primers
2.2. Construction of E. coli Genomic Library
2.3. Limit Screening Method (LMS Method) Combined with Tolerance for Gene Isolation
2.4. DNA Sequencing and Analysis
2.5. Construction of Plasmids
2.6. Promoter Replacement Based on I-SceI System
2.7. Pinene biosynthesis in shake flasks
2.8. GC analysis
2.9. Statistical analysis
3. Results and Discussion
3.1. Isolation of Genes Conferring Increased Pinene Accumulation in E. coli Using the LMS Method
3.2. Effect of I-SceI-Induced Recombination Promoter Replacement on Pinene Production
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strains/Plasmids | Description | Source/Purpose |
---|---|---|
Strain | ||
E. coli DH5α | supE44 Δ(lacZYA-argF) U169 (Φ80lacZ ΔM15) hsdR17 recA endA1 gyrA96 thi-1 relA1 | Invitrogen |
E. coli MG1655 | F- λ- ilvG- rfb-50 rph-1 | ATCC#700926 |
E. coli MG1655B | E. coli MG1655, P37-acrB | This study |
E. coli MG1655BF | E. coli MG1655B, P37-flgFG | This study |
E. coli MG1655BFN | E. coli MG1655BF, P37-ndk | This study |
Plasmid | ||
pMEVIGPS | pBbA5c-MTSAe-T1f-MBI(f)-T1002i-trGPPSA.grandis-PSA.grandi coding for MEV pathway enzymes to produce pinene from glucose in E. coli, p15A ori, PlacUV5 promoter, CmR | [10] |
pZEABP | Expression vector, P37 promoter, pBR322 ori, AmpR, BglBrick, ePathBrick contains four isocaudamer (AvrII, NheI, SpeI, and XbaI) | [29] |
pZEA-acrB | pZEA*BP contains acrB of E. coli, P37 promoter, pBR322 ori, AmpR | This study |
pZEA-flgFG | pZEA*BP contains flgFG of E. coli, P37 promoter, pBR322 ori, AmpR | This study |
pZEA-motB | pZEA*BP contains motB of E. coli, P37 promoter, pBR322 ori, AmpR | This study |
pZEA-ndk | pZEA*BP contains ndk of E. coli, P37 promoter, pBR322 ori, AmpR | This study |
pZEA-pbpc | pZEA*BP contains pbpc of E. coli, P37 promoter, pBR322 ori, AmpR | This study |
pUC57 | T vector, pBR322 ori, AmpR | Addgene |
pUC57-P37-S-Kan-S-P37 | pUC57 contains P37-S-Kan-S-P37 cassette | This study |
pUC-IsecI-acrB | pUC57-P37-S-Kan-S-P37 containing upstream and downstream homologous arms for promoter replacement of acrB | This study |
pUC-IsecI-flgFG | pUC57-P37-S-Kan-S-P37 containing upstream and downstream homologous arms for promoter replacement of flgF | This study |
pUC-IsecI-ndk | pUC57-P37-S-Kan-S-P37 containing upstream and downstream homologous arms for promoter replacement of ndk | This study |
pSIMIS | pSC101 repliconts PL-gam-bet-exo cI857, arabinose-inducible I-SceI endonuclease gene, AmpR | [30] |
Positive Clone | Gene | Protein | Reference |
---|---|---|---|
P1 | pbpc-ndk | peptidoglycan glycosyltransferase PbpC, nucleoside diphosphate kinase | [33,34] |
P2 | flgF-flgG | flagellar basal-body rod protein FlgF, flagellar basal-body rod protein FlgG | [35,36] |
P3 | motB | protein that enables flagellar motor rotation | [37] |
P4 | acrB | multidrug efflux pump RND permease AcrB | [38] |
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Huang, M.-Y.; Wang, W.-Y.; Liang, Z.-Z.; Huang, Y.-C.; Yi, Y.; Niu, F.-X. Enhancing the Production of Pinene in Escherichia coli by Using a Combination of Shotgun, Product-Tolerance and I-SceI Cleavage Systems. Biology 2022, 11, 1484. https://doi.org/10.3390/biology11101484
Huang M-Y, Wang W-Y, Liang Z-Z, Huang Y-C, Yi Y, Niu F-X. Enhancing the Production of Pinene in Escherichia coli by Using a Combination of Shotgun, Product-Tolerance and I-SceI Cleavage Systems. Biology. 2022; 11(10):1484. https://doi.org/10.3390/biology11101484
Chicago/Turabian StyleHuang, Ming-Yue, Wei-Yang Wang, Zhen-Zhen Liang, Yu-Chen Huang, Yi Yi, and Fu-Xing Niu. 2022. "Enhancing the Production of Pinene in Escherichia coli by Using a Combination of Shotgun, Product-Tolerance and I-SceI Cleavage Systems" Biology 11, no. 10: 1484. https://doi.org/10.3390/biology11101484
APA StyleHuang, M. -Y., Wang, W. -Y., Liang, Z. -Z., Huang, Y. -C., Yi, Y., & Niu, F. -X. (2022). Enhancing the Production of Pinene in Escherichia coli by Using a Combination of Shotgun, Product-Tolerance and I-SceI Cleavage Systems. Biology, 11(10), 1484. https://doi.org/10.3390/biology11101484