Part:BBa_K4759235
T7-RBS1-petH-RBS2-petF(Q62E)
Design
Redox proteins and redox proteases are linked by RBS2, joining the two proteins together. The original base of the amino acid encoding PetF at position 62 is CAG, we mutated it to GAG.
Usage and Biology
After obtaining the best redox partners PetH/PetF, we performed alanine scanning on petF to speculate which sites had a greater impact on its electron transport capacity. Finally, we found that after mutations in seven of them, the electron transport effect would change greatly, so we mutated the amino acids of these sites into other 19 amino acids by modeling and selecting 23 of them to get better results. (Site-directed Mutagenesis parts can be searched from BBa_K4759053 to BBa_K4759075, and constructed modeling screening for redox partners can be searched from BBa_K4759076 to BBa_K4759099)
Fig. 1: Fermentation of 23 mutants and control groups
We conducted control tests with the positive control group, negative control group, and wild-type strains. Finally, we selected 9 mutants with the highest fluorescence intensity for subsequent catalytic verification by detecting their green fluorescence intensity.
Fig. 2: 9 mutants + wild-type + negative control, 50 ml/250 ml system fermentation
By verifying the catalytic ability, we found that the substrate conversion of D68P was higher than that of the wild type in the nine strains with high fluorescence intensity, reaching 89.2%.
Fig. 3: A:Fluorescence intensity of wild type with 23 mutants B: Conversion of the 9 mutants with the highest fluorescence intensity with wild type
References
[1] Li, S.; Du, L.; Bernhardt, R. Redox Partners: Function Modulators of Bacterial P450 Enzymes. Trends Microbiol 2020, 28, 445-454. DOI: 10.1016/j.tim.2020.02.012
[2] Lou, D.; Tan, J.; Zhu, L.; Ji, S.; Tang, S.; Yao, K.; Han, J.; Wang, B. Engineering Clostridium absonum 7alpha-hydroxysteroid Dehydrogenase for Enhancing Thermostability Based on Flexible Site and DeltaDeltaG Prediction. Protein Pept Lett 2018, 25, 230-235. DOI: 10.2174/0929866524666171113113100
[3] Sagadin, T.; Riehm, J.; Putkaradze, N.; Hutter, M. C.; Bernhardt, R. Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding. FEBS J 2019, 286, 1240-1249. DOI: 10.1111/febs.14722
[4] Meng, S.; Li, Z.; Ji, Y.; Ruff, A. J.; Liu, L.; Davari, M. D.; Schwaneberg, U. Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s. Chinese Journal of Catalysis 2023, 49, 81-90. DOI: 10.1016/s1872-2067(23)64445-6
[5] Tovchigrechko, A.; Vakser, I. A. GRAMM-X public web server for protein-protein docking. Nucleic Acids Res 2006, 34, W310-314. DOI: 10.1093/nar/gkl206
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1342
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1342
Illegal NotI site found at 1115 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1342
Illegal BglII site found at 1653
Illegal BamHI site found at 1336 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1342
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1342
- 1000COMPATIBLE WITH RFC[1000]
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