Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade
Abstract
:1. Introduction
2. Results and Discussion
2.1. Cloning and Expression of Promiscuous Kinases
2.2. Characterization of Recombinant Promiscuous Kinases
2.3. Cloning and Expression of Isopentenyl Phosphate Kinase
2.4. Characterization of Recombinant Isopentenyl Phosphate Kinase
2.5. Developing a One-Pot Enzymatic Cascade for the Synthesis of DMAPP
2.6. Optimizing the Conditions of the One-Pot Enzymatic Cascade
3. Materials and Methods
3.1. Strains, Plasmids, Media and Chemicals
3.2. Plasmid Construction
3.3. Heterologous Expression and Purification of Recombinant Enzymes
3.4. Promiscuous Kinase Assay
3.5. Isopentenyl Phosphate Kinase Assay
3.6. Determination of Enzyme Activities and Properties
3.7. Optimizing the Conditions of the Biocatalytic Cascade to Produce DMAPP
3.8. HPLC and Liquid Chromatography–Mass Spectrometry (LC–MS) Analyses
3.9. Statistical Analysis
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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Enzyme | Step | Total Protein (mg) | Total Activity (U) | Specific Activity (U/mg) | Yield (%) |
---|---|---|---|---|---|
SfPK | Cell extract | 478 | 15.3 | 0.032 | 100 |
Ni-NTA affinity | 56 | 10.7 | 0.19 | 69.9 | |
EcPK | Cell extract | 452 | 2.3 | 0.005 | 100 |
Ni-NTA affinity | 13 | 1.3 | 0.1 | 56.6 | |
ScPK | Cell extract | 278 | 18.1 | 0.065 | 100 |
Ni-NTA affinity | 18 | 8.5 | 0.47 | 46.9 | |
MtIPK | Cell extract | 489 | 2.7 | 0.0056 | 100 |
Ni-NTA affinity | 4 | 0.18 | 0.045 | 6.6 | |
MthIPK | Cell extract | 463 | 0.5 | 0.001 | 100 |
Ni-NTA affinity | 21 | 0.34 | 0.016 | 68 |
Cation | Residual Activity (%) | ||
---|---|---|---|
SfPK | EcPK | ScPK | |
Control | 100 ± 0.8 | 100 ± 0.4 | 100 ± 0.8 |
Fe3+ | 46.8 ± 2.1 | 5.8 ± 0.5 | 32.9 ± 1.1 |
K+ | 113.7 ± 1.2 | 79.1 ± 1.5 | 89.6 ± 0.8 |
Ba2+ | 138.9 ± 3.1 | 46.9 ± 0.9 | 56.2 ± 2.6 |
Sr2+ | 122.1 ± 1.6 | 66.6 ± 1.2 | 63.9 ± 1.9 |
Ca2+ | 99.4 ± 0.4 | 64.3 ± 0.6 | 96.4 ± 0.4 |
Cu2+ | 102.1 ± 0.8 | 68.4 ± 1.8 | 79.2 ± 0.8 |
Ni2+ | 32.5 ± 1.4 | 87.5 ± 1.1 | 64.6 ± 0.7 |
Co2+ | 105.7 ± 0.8 | 84.5 ± 0.8 | 33.2 ± 1.5 |
Zn2+ | 32.5 ± 1.0 | 44.6 ± 1.2 | 70.7 ± 1.8 |
Mn2+ | 90.0 ± 1.1 | 79.8 ± 0.5 | 81.2 ± 1.6 |
Na+ | 110.5 ± 0.5 | 81.5 ± 1.1 | 106.9 ± 0.6 |
Fe2+ | 80.5 ± 0.3 | 34.9 ± 0.9 | 52.6 ± 1.5 |
Li+ | 102.1 ± 0.9 | 81.5 ± 1.3 | 62.8 ± 0.9 |
Mg2+ | 182.9 ± 1.9 | 88.1 ± 1.2 | 113.8 ± 0.6 |
Al3+ | 152.5 ± 1.4 | 86.3 ± 1.4 | 77.2 ± 1.4 |
NH4+ | 118.5 ± 0.7 | 82.6 ± 0.8 | 73.5 ± 1.2 |
EDTA | 96.3 ± 0.9 | 48.1 ± 0.9 | 46.1 ± 0.5 |
Cation | Residual Activity (%) | |
---|---|---|
MtIPK | MthIPK | |
Control | 100 ± 0.9 | 100 ± 1.1 |
Fe3+ | 82.9 ± 1.1 | 101.6 ± 0.3 |
K+ | 44.6 ± 0.5 | 104.2 ± 1.0 |
Ba2+ | 96.0 ± 0.8 | 109.8 ± 0.7 |
Sr2+ | 41.1 ± 0.2 | 87.0 ± 0.9 |
Ca2+ | 104.0 ± 1.3 | 118.1 ± 1.5 |
Cu2+ | 77.7 ± 0.8 | 105.7 ± 0.8 |
Ni2+ | 86.3 ± 1.2 | 109.8 ± 1.2 |
Co2+ | 46.9 ± 1.6 | 102.6 ± 0.6 |
Zn2+ | 84.0 ± 0.5 | 106.2 ± 0.5 |
Mn2+ | 95.4 ± 1.8 | 103.6 ± 0.4 |
Na+ | 77.1 ± 1.1 | 105.7 ± 1.4 |
Fe2+ | 58.9 ± 0.7 | 104.7 ± 0.8 |
Li+ | 61.1 ± 0.4 | 105.2 ± 0.6 |
Mg2+ | 101.7 ± 1.8 | 116.6 ± 1.3 |
Al3+ | 76.0 ± 0.6 | 108.8 ± 0.5 |
NH4+ | 66.3 ± 0.7 | 90.2 ± 0.7 |
EDTA | 29.1 ± 1.6 | 46.1 ± 1.0 |
Plasmid/Strain | Description | References |
---|---|---|
Plasmids | ||
pCDFDuet-SfPK | pCDFDuet-1 carrying SfPK from Shigella flexneri. T7 promoter, CloDF13 ori; streptomycinr | This study |
pCDFDuet-EcPK | pCDFDuet-1 carrying EcPK from Escherichia coli str. K-12 substr. MG1655. T7 promoter, CloDF13 ori; streptomycinr | This study |
pET-28a-ScPK | pET-28a (+) carrying ScPK from Saccharomyces cerevisiae. T7 promoter, pBR322 ori; kanamycinr | This study |
pCDFDuet-MtIPK | pCDFDuet-1 carrying MtIPK from Methanolobus tindarius. T7 promoter, CloDF13 ori; streptomycinr | This study |
pCDFDuet-MthIPK | pCDFDuet-1 carrying MthIPK from Methanothermobacter thermautotrophicus str. Delta H. T7 promoter, CloDF13 ori; streptomycinr | This study |
pET-28a-AtIPK | pET-28a (+) carrying AtIPK from Arabidopsis thaliana. T7 promoter, pBR322 ori; kanamycinr | This study |
pETDuet-7-DMATS | pETDuet-1 carrying 7-DMATS from Aspergillus fumigatus. T7 promotor; ColE1 ori; ampicillinr | This study |
pG-KJE8 | Carrying chaperone protein DnaK gene (dnaK), chaperone protein DnaJ (dnaJ), chaperone protein GrpE gene (grpE), chaperone protein GroES gene (groES), and chaperone protein GroEL gene (groEL); dnaK, dnaJ, and grpE were promoted by the araB promotor; grpES and groEL were promoted by the tetR promoter | TaKaRa Bio a |
pGro7 | Carrying the groES and groEL genes; groES and groEL were promoted by the araB promoter | TaKaRa Bio |
pKJE7 | Carrying the dnaK, dnaJ, and grpE genes; dnaK, dnaJ, and grpE were promoted by the araB promoter | TaKaRa Bio |
pTf16 | Carrying the trigger factor gene tig; tig was promoted by the araB promoter | TaKaRa Bio |
Strains | Escherichia coli BL21 (DE3) | New England Biolabs b |
BL21-SfPK | BL21 (DE3) harboring pCDFDuet-SfPK | This study |
BL21-EcPK | BL21 (DE3) harboring pCDFDuet-EcPK | This study |
BL21-ScPK | BL21 (DE3) harboring pET-28a-ScPK | This study |
BL21-MtIPK | BL21 (DE3) harboring pCDFDuet-MtIPK | This study |
BL21-MthIPK | BL21 (DE3) harboring pCDFDuet-MthIPK | This study |
BL21-AtIPK | BL21 (DE3) harboring pET-28a-AtIPK | This study |
BL21-7-DMATS | BL21 (DE3) harboring pETDuet-7-DMATS | This study |
BL21-AtIPK- KJE8 | BL21 (DE3) harboring pET-28a-AtIPK and pG-KJE8 | This study |
BL21-AtIPK-pGro7 | BL21 (DE3) harboring pET-28a-AtIPK and pGro7 | This study |
BL21-AtIPK-pKJE7 | BL21 (DE3) harboring pET-28a-AtIPK and pKJE7 | This study |
BL21-AtIPK-pTf16 | BL21 (DE3) harboring pET-28a-AtIPK and pTf16 | This study |
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Qiu, C.; Liu, Y.; Wu, Y.; Zhao, L.; Pei, J. Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade. Int. J. Mol. Sci. 2022, 23, 12904. https://doi.org/10.3390/ijms232112904
Qiu C, Liu Y, Wu Y, Zhao L, Pei J. Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade. International Journal of Molecular Sciences. 2022; 23(21):12904. https://doi.org/10.3390/ijms232112904
Chicago/Turabian StyleQiu, Cong, Yang Liu, Yangbao Wu, Linguo Zhao, and Jianjun Pei. 2022. "Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade" International Journal of Molecular Sciences 23, no. 21: 12904. https://doi.org/10.3390/ijms232112904