Part:BBa_K4759077

petH-RBS2-petF(D21F)-linker-GFP1-10

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.

Usage and Biology

Generally, the method of determining whether the redox partners was suitable required tedious steps such as the construction of plasmids, heterologous expression, construction of catalytic systems, and detection of conversion rate after catalysis. Therefore, we wanted to find a convenient way to do a quick screening. We used the fluorescent protein sfGFP and successfully constructed a sensor to detect redox partners. We divided sfGFP into N-terminal and C-terminal, and although these two parts were cut off, there was an interaction force between them. Thus, redox proteins were fused to the N-terminal of sfGFP-1-10 and Olep to the C-terminal of sfGFP-11, respectively.

Fig. 1: The self-assembly of Olep and Fdx based on the three-dimensional structure of sfGFP (PDB: 5BT0)

After obtaining the best redox partner, 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 show better results.

Fig2: Fermentation of 23 mutants and control groups 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 BglII site found at 2229
  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]