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Applications of BBa_K4759043

The P450 enzyme requires electrons (NADH, NADPH) to function. Electrons are first transferred to FDR (ferroxyreductase), to FDX

(ferro-reducing protein) under the action of ferroxyreductase, and then to P450 enzyme under the action of redox partner, and

finally to the heme center, after which the enzyme can react with the substrate and undergo hydroxylation.

Through extensive reading of the literature, we summarized 11 pairs of redox partners with good results. After that, we screened

four pairs of redox partners with good effects by molecular docking and mathematical modeling and then verified them

experimentally.

Generally, the method of determining whether the redox partner is suitable is through 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 to successfully construct a sensor to detect redox partners. sfGFP is a superfolder fluorescent protein that emits green light when irradiated with ultraviolet light. What is special about it is that it can be broken into two parts. We divide sfGFP into sfGFP-1-10 and sfGFP-11, and although these two parts are cut off, there is an interaction force between them, and as long as they are properly folded in space, they will emit light again. Thus, four iron redox proteins are fused to the N-terminus of sfGFP-1-10 and Olep to the C-terminus of sfGFP-11, respectively, to obtain the recombinant plasmid pRSFDuet-BM3-GFP-1-10-GFP-11-oleP, pRSFDuet-camA-camB-GFP-1-10-GFP-11-oleP, pRSFDuet-FdR_0978-Fdx_1499-GFP-1-10-GFP-11-oleP, pRSFDuet-petH-petF-GFP-1-10-GFP-11-oleP The above four recombinant plasmids are converted to BL21(DE3) to obtain recombinant strains G2 strain to G5 strain.

Fig1: The scheme of constructing sfGFP sensor

Fig2: The self-assembly of Olep and Fdx based on the three-dimensional structure of sfGFP (PDB: 5BT0)

The recombinant strains G2 to G5 are subjected to shaker fermentation experiments. After the fermentation is completed, 200 ul bacteria are added to the 96-well plate with a microplate reader to determine biomass (wavelength 600 nm) and fluorescence value (excitation wavelength 488 nm, emission wavelength 520 nm). Calculate the fluorescence intensity (fluorescence value/biomass) of the strain. The fluorescence intensity of the recombinant strain G5 (containing recombinant plasmid pRSFDuet-petH-petF-GFP-1-10-GFP-11-olep) is the highest (1.2×106) and 6 times higher than that of the control strain G2 (containing recombinant plasmid pRSFDuet-camA-camB-GFP-1-10-GFP-11-olep).

We selected four conventional redox partners (BM3, CamA/CamB, SelFdR_0978/SelFdx_1499, petH/petF) in combination with the P450 enzyme. 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.

Fig3: (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

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