This is an enzyme involved in the heme biosynthesis pathway. Also known as uroporphyrinogen III synthase.
In our project, we divided the synthesis pathway of heme into two parts, one was the synthesis pathway of heme precursor ALA (upstream), and the other part was the synthesis pathway of ALA to heme (downstream).
For the upstream pathway, there were two pathways from glutamate to ALA, one was the C4 pathway and the other was the C5 pathway. The C4 pathway had been enhanced by the existing team, and the experimental results showed that the effect was not as good as the C5 pathway, so we chose the C5 pathway for modification. The C5 pathway had two key genes, one was hemA and the other was hemL. So we constructed plasmids containing hemA and hemL, and overexpressed them by transforming recombinant plasmids to E. coli.
The downstream pathway, that was, the synthesis pathway of heme, involved 7 genes, of which 4 genes ( hemB, hemD, hemC, hemH ) were more critical, according to the literature. Therefore, we also constructed and overexpressed plasmids containing hemB, hemD, hemC, and hemH.

Fig. 1-1: Heme biosynthetic pathways in E. coli. The purple arrow represents the C5 pathway and the pink arrow represents the downstream biosynthetic pathway of heme. The pCDFDuet-hemA-hemL plasmid was constructed to enhance the C5 pathway; the pETDuet-hemBDC-hemH plasmid was constructed to enhance the downstream biosynthetic pathway

Therefore, 3 recombinant strains were constructed, E. coli AL strain(The recombinant plasmid pCDFDuet-hemLhemA-hemL was expressed in E. coli O2), E. coli BCDH strain (The recombinant plasmid pETDuet-hemB-hemC-hemD-hemH was expressed in E. coli O2), E. coli AL-BCDH strain(The recombinant plasmids pCDFDuet-hemA-hemL and pETDuet-hemB-hemC-hemD-hemH were expressed in E. coli O2).

hemA (BBa_K4759020) and hemL (BBa_K4759025) were expressed in BBa_K4759210, hemH (BBa_K4759024) was expressed in BBa_K4759211. hemB (BBa_K4759021) /hemC (BBa_K4759022) /hemD (BBa_I716155) were expressed in BBa_K4759270.

E. coli O2:

P450 enzyme CYP107D1 (Olep), derived from Streptomyces antibioticus, was synthesized after codon optimization (BBa_K4759001). The oleP expression gene, codon-optimized ferredoxin reductase gene camA, and ferredoxin gene camB were then subcloned to the plasmid pRSFDuet to obtain the recombinant plasmid pRSFDuet-camA-camB-olep.

In the plasmid pRSFDuet, Olep was expressed in BBa_K4759203 and CamA/CamB was expressed in BBa_K4759201.

The expression host was E.coli C41 (DE3), induction temperature was 25°C, and the inducer IPTG concentration was 0.5 mM.

After the end of induction, 1 mL of fermentation broth was collected and the cells were harvested by centrifugation at 14,000 rpm for 10 min at 4 °C. 0.2 mL of the supernatant was taken and mixed with 0.2 mL of ammonia and 1.6 mL of acetonitrile. Then centrifuged it at 14,000 rpm for 20 min, collected the supernatant, which was used to detect extracellular heme. Resuspended the pellet with 0.2 mL of ultrapure water, 0.2 mL of ammonia, and 1.6 mL of acetonitrile. The samples were then placed on ice and the cell suspension was sonicated with an ultrasonic disruptor for 10 min (power 30 W, 3 mm conical microtip probe with a pulse period of 3 s on/3 s off). The supernatant were collected by centrifugation at 14,000 rpm for 20 min, and it was used to detect intracellular heme.
Heme concentration was measured by UHPLC-QTOF-MS (Agilent 1290-6495C).

We managed to get the following data: Without the addition of ALA and FeCl₃, the heme binding rate of E. coli AL-BCDH strain was 27.86%, and the conversion rate was 5.83%; The heme binding rate of E. coli BCDH strain was 38.78%, and the conversion rate was 11.36%.

Fig. 1-2: The effect of enhancing heme biosynthesis on the Olep catalysis The blue-filled triangle represents the heme-binding ratio (%). The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates.

BBa_K339000: New data on maltose-binding protein (MBP)

Because the solubility-enhancing tag can promote the refolding of the target protein, we fused six protein tags (SUMO, GST, MBP, TF, Trx, Nus) at the N-terminal of the Olep.

We found protein tags MBP and TF can significantly improve the solubility of Olep.
However, the catalytic efficiency of Olep was reduced. Compared with the analytic label Olep (32.4%), the conversion rate of MBP-Olep and TF-Olep was only 27.53% and 8.49%, respectively .

Fig. 2-1: The application of protein tags The blue-filled triangle represents the biomass (OD₆₀₀). The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates.

After the soluble optimization, compared with the E. coli O2 strain (67.1%), the solubility of MBP-Olep reached 92.3% while TF-Olep reached 91.5%.
However, when measuring the heme-binding rate, it was found that the heme binding rate of MBP-Olep and TF-Olep was only 21.1% and 16.8% , which was lower than that of the heme binding rate of the analytic label Olep (39.4%). This might explain why the conversion rate of MBP-Olep and TF-Olep was only 27.53% and 8.49%, respectfully.
Therefore, we found insufficient heme supply may be a key factor limiting the catalytic efficiency of Olep.

Fig. 2-2: The heme-binding ratio of wild-type Olep, MBP-Olep and TF-Olep The blue-filled triangle represents the heme-binding ratio (%). The red hollow triangle represents the conversion rate (%). Values and triangles represent the means and standard deviations of biological triplicates.

We believe these information about BBa_K339000 will be helpful for future teams.

Redox patner Sensor

The P450 enzyme requires electrons (NADH, NADPH) to function. Electrons are first transferred to FDR (ferredoxin reductase), to FDX (ferro-reducing protein), and then to P450 enzyme under the action of redox partners, and finally to the heme center, after which the enzyme can react with the substrate and undergo hydroxylation.
Generally, the method of determining whether the redox partners was suitable required 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 and successfully constructed a sensor to detect redox partners!
You can see the detailed information in our Result page.
We believe that the construction of this sensor can provide reference and help for future teams studying P450 enzymes to screen suitable redox partners!

Fig. 3-1: Strategies to construct sfGFP sensor to screen redox partners. (A) The scheme of constructing sfGFP sensor. (B) The self-assembly of Olep and Fdx based on the three-dimensional structure of sfGFP (PDB: 5BT0). (C) 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. (D) 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.