Part:BBa_K4863000

α-carbonic anhydrase from Helicobacter pylori 26695 (hpCA)

A-carbonic anhydrases are metalloenzymes responsible for catalyzing the interconversion of carbon dioxide (CO2) to bicarbonate ions (HCO3−). hpCA is an α-carbonic anhydrase isolated from the bacterium Helicobacter pylori 26695, codon optimized for expression in E. Coli BL21(DE3) (Zhu et al., 2022).

In our work, CA is expressed in E. Coli and purified for display on the surface of Synechocystis PCC6803 for converting carbon dioxide into bicarbonate ions, which then forms calcium carbonate (CaCO3) precipitates, thus achieving biomineralization and the production of biological concrete.

Usage and Biology

Carbonic anhydrases (CAs) can catalyze the biochemical process necessary for the biotechnological method of microbially induced calcite precipitation (MICP), which has gained increasing attention due to its potential in creating calcium carbonate precipitation for increasing the compressive strength of concrete. CAs catalyze the conversion of carbon dioxide to bicarbonate ions, and the ions can react with CaCl to form calcium carbonate deposition, which acts as an adhesive for increasing and maintaining compressive strength of concrete. hpCA, the α-carbonic anhydrase isolated from Helicobacter pylori strain 26695, has relatively high performance among the CAs characterized so far for expression in E. Coli. Additionally, it has been shown that surface display is necessary for maintaining the stability of the enzyme (Zhu et al., 2022).

A-CAs are naturally present in mammals and many Eubacteria species and is responsible for various biochemical processes including and not limiting to maintaining concentration of carbon dioxide or bicarbonate ions for carboxylation reactions in autotrophs, transporting carbon dioxide or bicarbonate ions in mammals, and regulating intracellular pH(Bury-Moné, 2008). HpCA is encoded by the open reading frame HP1186 in Helicobacter pylori 26695. It is attached to the cell surface and is responsible for creating a CO2-rich environment which is necessary for survival of the organism(Chirica, 2002).

Source

Helicobacter pylori 26695

Characterization

The expression of hpCA in E. coli BL21 (DE3) is under the control of IPTG induction system. On plasmid pKeystone014, a lac repressor and a lac operon are installed to regulate the gene expression with response to IPTG supply. In our later tests of hpCA, the IPTG induction system helps us to verify the successful expression of the enzyme by E. coli.

We transformed the plasmid into E. coli DH5𝛼 for plasmid amplification in large quantities. The gel electrophoresis of the colony PCR product of the transformed strain indicates successful construction of the plasmid, which is also confirmed by the sequencing result.

Fig.1 Gel electrophoresis verification of the hpCA sequence after plasmid transformation into E. coli DH5𝛼. The primers used for colony PCR correspond to a 993bp-long product, which is close to the 1000bp bands observed.

The plasmid pKesytone014 was constructed via Gibson assembly of the different genetic parts. The T7 promoter was originally included in the pET plasmid backbone (LZ002-pET28a-NT2RepCT-BGI) along with the lac repressor gene. The hpCA sequence was artificially synthesized and then inserted into the plasmid backbone to form the complete pKeystone014 plasmid.

We transformed the plasmid into E. coli DH5𝛼 for plasmid amplification in large quantities. The gel electrophoresis of the colony PCR product of the transformed strain indicates successful construction of the plasmid, which is also confirmed by the sequencing result.

After plasmid amplification in E. coli DH5𝛼, we extracted the plasmids from the strain and transformed them into E. coli BL21 (DE3) for hpCA expression. Due to the IPTG induction system engineered on the plasmid pKeystone014, only the bacteria treated with IPTG during culture can activate the expression of hpCA. This is reflected in our SDS-PAGE result of the protein purified from E. coli BL21 (DE3) expressing the enzyme.

Fig.2: SDS-PAGE verification of extracted hpCA protein. The plasmid we built for hpCA expression in E. coli utilizes IPTG induction to achieve gene expression control. The lane on the left labelled “IPTG-” corresponds to protein extracted from E. coli strain without IPTG added during culture, whereas the lane on the right labelled “IPTG+” corresponds to protein extracted from E. coli strain with IPTG added during culture. hpCA has a molecular weight of 27 kDa; the appearance of the 30-kDa band indicated by the red arrow is close to the molecular weight of hpCA, verifying the successful expression of hpCA by IPTG+ strain.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 414
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

Bury-Moné, S., Mendz, G. L., Ball, G. E., Thibonnier, M., Stingl, K., Ecobichon, C., Avé, P., Huerre, M., Labigne, A., Thiberge, J.-M., & DeReuse, H. (2008). Roles of α and β carbonic anhydrases of Helicobacter pylori in the urease-dependent response to acidity and in colonization of the murine gastric mucosa. Infection and Immunity, 76(2), 497-509. https://doi.org/10.1128/iai.00993-07

Chirica, L. C., Petersson, C., Hurtig, M., Jonsson, B.-H., Borén, T., & Lindskog, S. (2002). Expression and localization of α- and β-carbonic anhydrase in Helicobacter pylori. Biochimica Et Biophysica Acta (BBA) - Proteins and Proteomics, 1601(2), 192-199. https://doi.org/10.1016/s1570-9639(02)00467-3

Zhu, Y., Liu, Y., Ai, M., & Jia, X. (2022). Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralization. Synthetic and Systems Biotechnology, 7(1), 460-473. https://doi.org/10.1016/j.synbio.2021.11.008