Part:BBa_K4759061

petH-RBS2-petF(D61T)

Aspartic acid at site 61 of the PetF is mutated to threonine.

Usage and Biology

The P450 enzymes are redox-dependent proteins, through which they source electrons from reducing cofactors to drive their activities. This part is coding for ferredoxin reductase PetH and ferredoxin PetF from the algae (Synechocystis PCC 6803) as the redox chaperones of OleP.
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 selected 23 of them to get better results.

Fig1: Fluorescence intensity of wild type with 23 mutants

We conducted control tests with the positive control group, negative control group, and wild-type strains, and finally selected 9 mutants with the highest fluorescence intensity for subsequent catalytic verification by detecting their green fluorescence intensity.

Fig2: Fermentation of 23 mutants and control groups

We conducted control tests with the positive control group, negative control group, and wild-type strains, and finally selected 9 mutants with the highest fluorescence intensity for subsequent catalytic verification by detecting their green fluorescence intensity.

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%.

Fig3: Conversion of the 9 mutants with the highest fluorescence intensity with wild type

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1249
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1249
  Illegal NotI site found at 1022
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1249
  Illegal BglII site found at 1560
  Illegal BamHI site found at 1243
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1249
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1249
  
• 1000
  COMPATIBLE WITH RFC[1000]