Part:BBa_K4665005
Usage and Biology
Biomineralization is the process by which living organisms synthesise minerals (Dhami et al., 2013). Microbial calcium carbonate production can proceed through two main metabolic pathways, using urease or carbonic anhydrase (CA) as the catalysts of the reaction (Chaparro-Acuña et al., 2019). However, synthesis through urea hydrolysis produces toxic byproducts which is not observed in the CA catalyzed pathway.
SazCA, derived from the thermophilic bacterium Sulfurihydrogenibium azorense, is the fastest known carbonic anhydrase to date, boasting a kcat/KM value of 3.5 × 108 M−1 s−1 (De Simone et al, 2015). SazCA facilitates the hydration of carbon dioxide to bicarbonate and protons, creating alkaline conditions that aid the formation of calcium carbonate crystals on the extracellular matrix (EPS) of bacterial cells (Fig. 1) (Anbu, et al. 2016).
To enhance enzymatic efficiency, this composite part expresses the SazCA enzyme as a fusion protein on the cell surface of *E. coli*. This approach bypasses cellular limitations and directly exposes the enzyme to extracellular CO2, increasing calcium carbonate production on limestone surfaces.
This component is based on the findings of Zhu et al. (2022), wherein a membrane fusion protein was designed to showcase SazCA on the surface of *E. coli* cells. This is achieved by linking the E. coli codon-optimized SazCA enzyme (**BBa_K4665120**) to the integral membrane protein INPN (**BBa_K4665001**) using a flexible GGGGS linker (**BBa_K2549053**).
[This composite part consists of three basic parts:
1) Ice nucleation protein N-terminal (INPN): This is the N-terminal of ice nucleation protein which will be embedded into the E. coli cell membrane. The sequence coding for the INPN is preceded by a pelB leader sequence. By attaching the pelB signal peptide in front of the INP protein, the fusion protein will be directed towards the bacterial periplasm where it will be anchored in the cell membrane (Singh et al., 2013). The INPN sequence is followed by two front-end sub-repeat sequences important for the stability of the fusion protein (Zhu et al., 2022).
2) GGGGS linker: The GGGGS flexible linker is composed of a sequence of 4 glycine repeats followed by a serine amino acid. This flexible linker is used to connect the N-terminal of the INP to the carbonic anhydrase.
3) SazCA: This sequence codes for the carbonic anhydrase derived from Sulfurihydrogenibium azorense (SazCA). This sequence has been codon optimised for E. coli. The SazCA coding sequence is followed by a His-tag which facilitates the purification and detection of the fusion protein.
The recombinant carbonic anhydrase plasmid was constructed using a pET-39b(+) backbone vector which has several features that make it a suitable backbone for the recombinant plasmid. It includes a T7 promoter with a lac operator, enabling the selective expression of our fusion protein when incubated with Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Du et al., 2021). Additionally, the pET-39b(+) vector possesses a kanamycin resistance gene which does not contain any internal BsaI sites that might interfere with the assembly of the recombinant plasmid via Golden Gate Assembly. The kanamycin resistance gene allows for the positive selection of E. coli cells which have successfully incorporated the recombinant plasmid by their ability to grow on a growth medium infused with kanamycin.]
Characterisation
In Vitro Mineralization:
To test the ability of engineered BL21(DE3) E. coli strain to precipitate CaCO3, we performed an in vitro mineralisation assay, adapting Zhu, et al. 's technique. Bacteria were cultured overnight in 30mL of LB +Kanamycin medium and 0.5 mM ZnSO4 at 25℃. IPTG induction was performed 3 hours prior to experimentation. The assay was run on 8 mL Tris-HCl buffer 8.3 and 50mL of saturated CO2 aqueous solution at 0℃. 3 mL of cell pellet were introduced into the solution, and the reaction was allowed to proceed on ice for an hour. At this point, the bacteria should have been able to produce bicarbonate ions. Cells were removed from the solution by centrifugation (15 min x 5000g). 25mL of a 0.3M solution of CaCl2 was added to the remaining supernatant as a calcium source. The reaction was left to run at 25℃ for 12h. Samples were filtered using vacuum filtration and dried at 50℃ to evaporate the solvent. Solid mass was weighed and recorded as “Wet Weight”. Upon preliminary analysis of FT-IR data, it was concluded that the mineral sample contained a large amount of water, elucidated by the stretching O-H peak at 3400 cm.1. Hence, the sample was dried further in liquid nitrogen for 48 hours, final weight was recorded at 2.3 g (yield=306.17%). Precipitated dry crystals were analysed using ATR-IR and 3C NMR
References
Anbu, P. et al. (March 1, 2016). Formations of calcium carbonate minerals by bacteria and its multiple applications. Springerplus 5(250). https://doi.org/10.1186/s40064-016-1869-2
Chaparro-Acuña, S.P., et al. (June, 2018). Soil bacteria that precipitate calcium carbonate: mechanism and applications of the process. Acta Agronómica 67(2). https://doi.org/10.15446/acag.v67n2.66109
De Luca, V. et al. (March 15, 2013). An α-carbonic anhydrase from the thermophilic bacterium Sulphurihydrogenibium azorense is the fastest enzyme known for the CO2 hydration reaction. Bioorganic & Medicinal Chemistry Letters, 21(6): 1465.1469. https://doi.org/10.1016/j.bmc.2012.09.047
De Simone, G., et al. (May 1, 2015). Crystal structure of the most catalytically effective carbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense. Bioorganic & Medicinal Chemistry Letters, 1;25(9): 2002-2006. https://doi.org/10.1016/j.bmcl.2015.02.068
Dhami, N.K., et al. ( May 2013). Biomineralization of calcium carbonate polymorphs by the bacterial strains isolated from calcareous sites. Journal of Microbiology and Biotechnology, 23(5): 707-714. https://doi.org/10.4014/jmb.1212.11087
Jo, B.H. (October 3, 2013). Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration. Applied and Environmental Microbiology. https://doi.org/10.1128/AEM.02400-13
Pan, S. H., & Malcolm, B. A. (2000). Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3). BioTechniques, 29(6), 1234–1238. https://doi.org/10.2144/00296st03
Sequence and Features
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