Efficient Production of 9,22-Dihydroxy-23,24-bisnorchol-4-ene-3-one from Phytosterols by Modifying Multiple Genes in Mycobacterium fortuitum
Abstract
:1. Introduction
2. Results
2.1. Construction of Phylogenetic Tree of Hsd4A, FadA5, and OpccR
2.2. Hsd4A—The Key Enzyme in the C19 Pathway
2.3. Deletion of fadA5 Improves the Selectivity of 9-OHBA by Eliminating 9-OHAD
2.4. Evaluation of the 9-OHBA-Producing Strain
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains, Plasmids, Reagents, and Culture Conditions
4.2. Bioinformatic Analysis
4.3. Construction of Mutant Strains
Name | Description | Source |
---|---|---|
Strains | ||
Escherichia coli | E. coli DH5α | Vazyme Biotech Co., Ltd., Nanjing, China |
MFKD | 9-OHAD producer, kstD1&2&3&4&5 deletion mutant of ATCC 35855 | Our lab |
MFKD_opccR | ATCC 35855 opccR overexpression in MFKD via p40-opccR | This study |
MFKDΔhsd4A | hsd4A deletion mutant of MFKD | This study |
MFKDΔhsd4A_ opccR | ATCC 35855 opccR overexpression in MFKDΔhsd4A via p40-opccR | This study |
MFKDΔhsd4AΔfadA5 | hsd4A and fadA5 double-deletion mutant of MFKD | This study |
Plasmids | ||
pKADel | Plasmid for allelic exchange, Pag85-lacZ Phsp60-sacB, AprR, KanR | [19] |
pKADelΔhsd4A | pKADel carrying two homologous arms of hsd4A | This study |
pKADelΔfadA5 | pKADel carrying two homologous arms of fadA5 | This study |
p40 | pMV306 with Psmyc promoter, KanR | [19] |
p40-opccR | p40 possessing opccR from M. fortuitum ATCC 35855 | This study |
4.4. Bioconversion and Analytical Methods
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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Strain | Relative Selectivity (%) | |||
---|---|---|---|---|
9-OHAD | 9-OHBA | AD | BA | |
MFKD | 78.09 ± 0.21 | 4.21 ± 0.08 | 0.82 ± 0.01 | 0.09 ± 0.01 |
MFKD_opccR | 77.98 ± 2.06 | 4.76 ± 0.31 | 0.87 ± 0.16 | 0.12 ± 0.03 |
MFKDΔhsd4A | 4.07 ± 0.12 | 81.47 ± 0.04 | 0.35 ± 0.01 | 0.21 ± 0.01 |
MFKDΔhsd4A_ opccR | 4,67 ± 0.13 | 81.22 ± 0.37 | 0.63 ± 0.22 | 0.19 ± 0.01 |
MFKDΔhsd4AΔfadA5 | 0.90 ± 0.08 | 95.13 ± 0.46 | 0.37 ± 0.08 | 0.19 ± 0.02 |
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Han, S.; Liu, X.; He, B.; Zhai, X.; Yuan, C.; Li, Y.; Lin, W.; Wang, H.; Zhang, B. Efficient Production of 9,22-Dihydroxy-23,24-bisnorchol-4-ene-3-one from Phytosterols by Modifying Multiple Genes in Mycobacterium fortuitum. Int. J. Mol. Sci. 2024, 25, 3579. https://doi.org/10.3390/ijms25073579
Han S, Liu X, He B, Zhai X, Yuan C, Li Y, Lin W, Wang H, Zhang B. Efficient Production of 9,22-Dihydroxy-23,24-bisnorchol-4-ene-3-one from Phytosterols by Modifying Multiple Genes in Mycobacterium fortuitum. International Journal of Molecular Sciences. 2024; 25(7):3579. https://doi.org/10.3390/ijms25073579
Chicago/Turabian StyleHan, Suwan, Xiangcen Liu, Beiru He, Xinghui Zhai, Chenyang Yuan, Yixin Li, Weichao Lin, Haoyu Wang, and Baoguo Zhang. 2024. "Efficient Production of 9,22-Dihydroxy-23,24-bisnorchol-4-ene-3-one from Phytosterols by Modifying Multiple Genes in Mycobacterium fortuitum" International Journal of Molecular Sciences 25, no. 7: 3579. https://doi.org/10.3390/ijms25073579