Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Strains, Oligonucleotides and Culture Growth
2.3. Construction of the M. smegmatis Heterologous Strains
2.4. Steroid Biotransformation Assay
2.5. Steroid Extraction and Analyses
Time (min) | %A | %B | %C |
0 | 50 | 50 | 0 |
5 | 50 | 50 | 0 |
15 | 20 | 71 | 9 |
20 | 4 | 87 | 9 |
40 | 0 | 85 | 15 |
41 | 0 | 85 | 15 |
42 | 50 | 50 | 0 |
52 | 50 | 50 | 0 |
Time (min) | %A | %B | %C |
0 | 50 | 50 | 0 |
5 | 50 | 50 | 0 |
15 | 20 | 71 | 9 |
20 | 0 | 91 | 9 |
40 | 0 | 70 | 30 |
41 | 0 | 85 | 15 |
42 | 50 | 50 | 0 |
52 | 50 | 50 | 0 |
2.6. NMR Spectra
3. Results and Discussion
3.1. Production of 14αOH-AD in M. Smegmatis from Sterols in Single Fermentation Step
3.2. Production of 14αOH-ADD in M. smegmatis from Sterols in Single Fermentation Step
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Strain | Description | Reference |
---|---|---|
Mycolicibacterium smegmatis mc2 155 | ept−1, mutant mc26 | [42] |
Mycolicibacterium smegmatis mc2 155 MS6039 | M. smegmatis mc2 155 ΔMSMEG_6039 | [34] |
Mycolicibacterium smegmatis mc2 155 MS6039-5941 | M. smegmatis mc2 155 ΔMSMEG_6039-ΔMSMEG_5941 | [34] |
Escherichia coli DH10B | F−, mcrA, Δ(mrr-hsdRMS-mcrBC), f80ΔlacZDM15 ΔlacX74, deoR, recA1, endA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL, nupG, λ− | Invitrogen |
Plasmid | Description | Reference |
pMV261 | KmR, Mycobacterium expression vector, PHsp60 | [43] |
pMVFAN | KmR, synthetic operon FAN into pMV261 | This work |
Oligonucleotide | Sequence | Application |
pMV4260 F | TTGCCGTCACCCGGTGACC | pMV261 sequencing |
pMV4486 R | ATCACCGCGGCCATGATGG | pMV261 sequencing |
64795 EcoRIF | CCGGAATTCTGACCTGAGAGAAAGGGAGTGATAAATGGCACAACTCGACACGC | CPR64795 amplification |
64795 XbaIR | GCTCTAGATTATCATGACCAGACGTCTTCCTG | CPR64795 amplification |
103168 BglXbaMunF | GAAGATCTTCTAGACAATTGTGACCTGAGAGAAAGGGAGTGATAAATGGATCCCCAGACTGTCG | CYP103168 amplification |
103168 XhoR | CCGCTCGAGTTACTACACTACCACTCTCTTGAAAGC | CYP103168 amplification |
64795 F2 | AATCAGCATTGCTGGCTCC | FAN operon sequencing |
64795 F3 | CTCCAACTTCAAGCTTCCTTCG | FAN operon sequencing |
64795 F4 | AATACGTCGCTTTCGGTCTCG | FAN operon sequencing |
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Felpeto-Santero, C.; Galán, B.; García, J.L. Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis. Microorganisms 2021, 9, 1499. https://doi.org/10.3390/microorganisms9071499
Felpeto-Santero C, Galán B, García JL. Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis. Microorganisms. 2021; 9(7):1499. https://doi.org/10.3390/microorganisms9071499
Chicago/Turabian StyleFelpeto-Santero, Carmen, Beatriz Galán, and José Luis García. 2021. "Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis" Microorganisms 9, no. 7: 1499. https://doi.org/10.3390/microorganisms9071499