Part:BBa_K4759220
T7-RBS1-petH-linker-petF-linker-Olep
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. To better improve the interaction of PetH and PetF, we use linker to link PetH and PetF.
Usage and Biology
The fusion combination strategy of redox partners can help 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 adopted the following strategies and all recombinant plasmid were transformed to C41 (DE3), respectively: recombinant strains R6 (PetH, PetF, and Olep were fused by two linkers); recombinant strains R7 (petH and petF were fused by linker); recombinant strains R8 (PetH and PetF was constructed on pACYCDuet, while oleP expression gene was constructed on pRSFDuet); recombinant strains R9 (petH and petF were fused by a linker, and were constructed on pACYCDuet, while the oleP expression gene was constructed on pRSFDuet).
Fig. 1: 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.
Fig. 2: The conversion rate of DCA for R5-R9 strains. The blue-filled triangle represents the biomass (OD600). Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1109
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1638
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1696
Illegal AgeI site found at 1794 - 1000COMPATIBLE WITH RFC[1000]
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