Heterologous Overexpression of Cytochrome P450BM3 from Bacillus megaterium and Its Role in Gossypol Reduction
Abstract
:1. Introduction
2. Results
2.1. Heterologous Expression of Cytochrome P450BM3 and Optimization of Gossypol Degradation Conditions
2.1.1. Construction of Cloning and Expression Vectors
2.1.2. Optimization of Cytochrome P450BM3 Enzyme Expression
2.1.3. Optimization of Cytochrome P450BM3 Catalytic Conditions for Gossypol Degradation
2.2. Site-Directed Mutagenesis of Cytochrome P450BM3 and Comparative Analysis of Gossypol Degradation
2.2.1. Prediction of Catalytic Active Sites in Cytochrome P450BM3 for Gossypol Reduction
2.2.2. Site-Directed Mutagenesis of Cytochrome P450BM3
2.2.3. Comparison of Gossypol Degradation and Enzymatic Activity Between Cytochrome P450BM3 and Its Mutants
2.3. Changes in Gossypol Metabolites Catalyzed by Cytochrome P450BM3 and Its Mutants
Screening of Differential Metabolites
3. Discussion
3.1. Construction and Expression of Cytochrome P450BM3 in E. coli
3.2. Optimization of Cytochrome P450BM3-Catalyzed Gossypol Degradation Conditions
3.3. Comparison of Catalytic Efficiency Between Cytochrome P450BM3 and Its Mutants
3.4. Degradation Products and Pathways of Gossypol Catalyzed by Cytochrome P450BM3 and Its Mutants
4. Conclusions
5. Materials and Methods
5.1. General
5.2. Bacterial Strains, Plasmids, and DNA Techniques
5.3. Protein Expression and Purification
5.4. Cytochrome P450BM3 Enzyme Activity Measurements
5.5. Examination of the Catalytic Effect of Cytochrome P450BM3 on Gossypol
5.6. HPLC Determination of Gossypol
5.7. Site-Directed Mutagenesis of Cytochrome P450BM3
5.8. Comparative Analysis of Catalytic Effect on Gossypol and Metabolite Detection by Cytochrome P450BM3 Enzyme and Its Mutants
5.9. Data Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
FG | Free gossypol |
BG | Bound gossypol |
CYP | Cytochrome P450 |
UPLC-Q-TOF/MS | Ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry |
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Group | Item | Compound | m/z | RT (min) | Regulate |
---|---|---|---|---|---|
P450BM3 | 1 | C28H28O9 | 507.16 | 6.64 | Up |
2 | C27H2807 | 465.19 | 6.79 | Down | |
3 | C25H24O6 | 419.15 | 6.76 | Down | |
4 | C16H14O6 | 301.07 | 6.98 | Up | |
5 | C16H18N2 | 239.15 | 5.50 | Up | |
R162H | 1 | C28H30O7 | 477.19 | 6.96 | Down |
2 | C25H24O5 | 405.17 | 6.76 | Down | |
3 | C16H14O6 | 301.07 | 6.98 | Up | |
4 | C16H20N2O3 | 367.18 | 6.82 | Up | |
Q129H | 1 | C28H30O8 | 493.19 | 6.45 | Down |
2 | C28H28O8 | 493.19 | 6.76 | Down | |
3 | C27H28O9 | 497.18 | 6.25 | Down | |
4 | C27H28O7 | 465.20 | 6.12 | Down | |
5 | C26H26O6 | 435.18 | 6.67 | Down | |
6 | C20H26N2O2 | 349.18 | 9.33 | Down | |
7 | C29H26N2O4 | 457.20 | 6.54 | Down | |
8 | C16H20N2O2 | 273.16 | 5.57 | Up |
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Fan, W.; Cui, J.; Xu, T.; Xu, S.; Ainiwaer, Z.; Luo, Q.; Wang, C. Heterologous Overexpression of Cytochrome P450BM3 from Bacillus megaterium and Its Role in Gossypol Reduction. Toxins 2025, 17, 253. https://doi.org/10.3390/toxins17050253
Fan W, Cui J, Xu T, Xu S, Ainiwaer Z, Luo Q, Wang C. Heterologous Overexpression of Cytochrome P450BM3 from Bacillus megaterium and Its Role in Gossypol Reduction. Toxins. 2025; 17(5):253. https://doi.org/10.3390/toxins17050253
Chicago/Turabian StyleFan, Wenpeng, Jingjing Cui, Tongxiang Xu, Shiheng Xu, Zulibina Ainiwaer, Qiyu Luo, and Caidie Wang. 2025. "Heterologous Overexpression of Cytochrome P450BM3 from Bacillus megaterium and Its Role in Gossypol Reduction" Toxins 17, no. 5: 253. https://doi.org/10.3390/toxins17050253
APA StyleFan, W., Cui, J., Xu, T., Xu, S., Ainiwaer, Z., Luo, Q., & Wang, C. (2025). Heterologous Overexpression of Cytochrome P450BM3 from Bacillus megaterium and Its Role in Gossypol Reduction. Toxins, 17(5), 253. https://doi.org/10.3390/toxins17050253