Part:BBa_I716155

hemD

This is an enzyme involved in the heme biosynthetic pathway. Also called uroporphyrinogen III synthase.

IISER_Bhopal 2021

Usage and Biology

During heme biosynthesis, this enzyme catalyses the asymmetric cyclization of linear tetrapyrrole to form a uroporphyrinogen III isomer. At the catalytic site, hydroxymethylbilane use as a substrate gives uroporphyrinogen III[1]. Reports have identified that along with hemA and hemL, upregulation of hemD and hemF increases the 5-Aminolevulinic acid accumulation which is a promising source for cancer treatment[2].

References

1. Schubert, H. L., Phillips, J. D., Heroux, A., & Hill, C. P. (2008). Structure and Mechanistic Implications of a Uroporphyrinogen III Synthase−Product Complex. Biochemistry, 47(33), 8648–8655.

2. Zhang, J., Kang, Z., Chen, J., & Du, G. (2015). Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli. Scientific Reports, 5(1).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Jiangnan-China 2023

Usage and Biology

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.

Fig1: 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-hemA-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).

Fig2: The color of engineered strains and pure enzyme. 1: E. coli O1 strain; 2: E. coli O2 strain; 3: E. coli O2 strain cultivated with ALA and FeCl₃; 4: E. coli AL strain; 5: Olep enzyme purified from E. coli AL strain. The lower values represent the content of intracellular heme in different engineered strains

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

Fig3: 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.