Part:BBa_K4759034
camA-RBS2-camB
The P450 enzymes are redox-dependent proteins that source electrons from reducing cofactors to drive their activities. This part is coding for ferredoxin reductase camA and ferredoxin camB from Pseudomonas putida as the redox chaperones of Olep.
Usage and Biology
Four groups of redox partners are constructed on the high-copy plasmid pRSFDuet to obtain recombinant plasmids: pRSFDuet-BM3-olep, pRSFDuet-camA-camB-olep, pRSFDuet-FdR0978-Fdx1499-olep, and pRSFDuet-petH-petF-olep. and transformed to C41 (DE3) to obtain the recombinant strain R2 strain to R5 strain. The recombinant strains G2 to G5 are subjected to shaker fermentation experiments. The recombinant strain R5 (containing recombinant plasmid pRSFDuet-petH-petF-olep) has the highest conversion rate, significantly increasing from 41.4% to 85.6%. Therefore, the redox companion PetH/PetF derived from Synechocystis is successfully screened as the most suitable redox partner for the P450 enzyme Olep, and the construction of the sfGFP sensor is verified, which could efficiently and accurately screen the redox partner adapted by the P450 enzyme.
Fig1: (A) Screening proper redox partners for Olep from different sources. The G1 strain that contains the empty pRSFDuet-1 plasmid was used as a control. The fluorescent intensities were calculated and the color of cells and fluorescent images were presented for G2-G5 strains that express different redox partners-sfGFP-1-10 and sfGFP-11-Olep, respectively. (B) The conversion rates were calculated for R2-R5 strains that express different redox partners and Olep, respectively. The R1 strain that contains the empty pRSFDuet-1 plasmid was used as a control. The blue-filled triangle represents the fluorescent intensity/OD600. The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates
Fig2: The structures and interactions between Olep and Fdxs are presented. The key interacting residues in Olep-Fdx complexes are depicted as sticks and highlighted in yellow. Heme and substrates are displayed as sticks, colored in red and wheat, respectively. The Fe2S2 cluster is visualized as spheres. The distances (Å) between the iron–sulfur cluster and heme-iron are measured and indicated by dashed red lines. The interaction areas of Olep-Fdx are calculated by NovoPro (https://www.novopro.cn/). The numbers of hydrogen bonds and salt bridges are predicted by PDBePISA (https://www.ebi.ac.uk/pdbe/)
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1279
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1279
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1279
Illegal BamHI site found at 1273 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1279
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1279
Illegal AgeI site found at 37
Illegal AgeI site found at 331 - 1000COMPATIBLE WITH RFC[1000]
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