Difference between revisions of "Part:BBa K4759218"

Revision as of 16:24, 10 October 2023

T7-RBS1-RBM3

Design Notes

(BM3 Redox domain)：a gene coding for Reducatase domain from P450BM3(the selfsufficient P450 enzyme). it has an excellent electron transfer system and the fastest substrate oxidation rate among the P450s owing to the proximity and specific positioning of its domains . Researcher has found that fusing P450s to reductase genes can improve the biocatalysis, We use this part to construct electron transfer pathway to Olep

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