Part:BBa_K4759052

petH-linker-petF

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

The fusion combination strategy of redox partners helps to improve the catalytic activity of Olep. To further screen the optimal redox partner, we decided to make a fusion combination of petF\petH. We designed to take different linkages, and combinations between petF\petH and OleP. We adopted the following strategies and all recombinant plasmid were transformed to C41 (DE3), respectively: recombinant strains R6 (petH and petF was connected by linker, and was connected to Olep through Linker); recombinant strains R7 (petH and petF was connected by linker); recombinant strains R8 (petH\petF was constructed on pACYCDuet, while oleP expression gene was constructed on pRSFDuet). recombinant strains R9 (petH and petF were connected by a linker, and were constructed on pACYCDuet, while the oleP expression gene was constructed on pRSFDuet). Fig1: The fusion expression and different expressional ratio between Olep and PetH/PetF

Recombinant strains R6 to R9 were subjected to shake flask fermentation. HPLC assay for product generation. The transformation rate of recombinant strains R6 to R9 were lower than that of the recombinant strain R5.

Fig2: The conversion rate of DCA for R5-R9 strains. The blue-filled triangle represents the biomass (OD600).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

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

  Illegal BglII site found at 1551
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]