Coding

Part:BBa_K5117033

Designed by: Jenny Sauermann, Lilli Kratzer, Katrin Lehmann   Group: iGEM24_TU-Dresden   (2024-09-28)
Revision as of 19:51, 30 September 2024 by Jesa98 (Talk | contribs)


BhrPET-L2

This part contains the gene of the uncultured bacterium HR29 encoding a polyethylene terephthalate hydrolase (PETase, EC 3.1.1.101). It is a homologue of the leaf-branch compost cutinase (LCC) and codon optimized for Bacillus subtilis (Xi et al. 2021). This sequence does not include any secretory signal peptide.

Downstream of the coding sequence, a long flexible linker (L2) has been added encoding the amino acids (GGGGS)4.


BhrPET-L2 only served for design purposes of the TU Dresden iGEM 2024 Team and was required for the construction of composite parts (see Contribution).


Target organism: Bacillus subtilis

Main purpose of use: Gene expression and production of fusion proteins (especially for spore surface display)


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 495
    Illegal AgeI site found at 540
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, XbaI, SpeI, PstI and NotI were removed from the coding sequence (CDS). To make the part compatible with the Type IIS standard, BsaI and SapI sites were removed as well. This was achieved by codon exchange using the codon usage table of Bacillus subtilis (Codon Usage Database Kazusa).


BhrPET-L2 is designed to be fused to the N-terminus of another protein. Therefore, the coding sequence does not contain a stop codon. Moreover, different linkers between the fused target enzyme and following protein can be analyzed, as these proteins may affect the folding and stability of each other and, eventually, lead to misfolding and reduced activity. Whereas flexible linkers promote the movement of joined proteins and are usually composed of small amino acids (e.g. Gly, Ser, Thr), rigid linkers are usually applied to maintain a fixed distance between the domains (Chen et al. 2013).

Within the framework of the TU Dresden iGEM 2024 Team, three linkers have been tested: 1) A short flexible GA linker (L1) encoding the small amino acids Gly and Ala, 2) A long flexible linker (GGGGS)4 (L2) which is one of the most common flexible linkers consisting of Gly and Ser residues and 3) A rigid linker GGGEAAAKGGG (L3) in which the EAAAK motif results in the formation of an alpha helix providing high stability (Chen et al. 2013).

The part BhrPET-L2, documented in this page, contains the long flexible linker (GGGGS)4.


Enzyme characterization according to literature

In the study by Xi et al. (2021) titled "Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29", a polyethylene terephthalate (PET) hydrolase BhrPETase was heterologously overexpressed in B. subtilis and biochemically characterized (Xi et al. 2021).

The gene encoding BhrPETase was first discovered in a metagenomic study by Kato et al. (2018). Its sequence is homologous to that of the leaf-branch compost cutinase (LCC) variant (94 % sequence identity) (Xi et al. 2021).

In the work of Xi et al., a secretory expression of BhrPETase was performed in B. subtilis, which was improved by the development of the chaperone-overexpression mutant strain. After protein overexpression, supernatants were collected and subsequently purified with hydrophobic interaction chromatography (HIC) followed by ion exchange chromatography (IEC). The collected fractions from each purification step were analyzed with SDS-PAGE and a purified protein band corresponding to the expected molecular weight of 27.5 kDa was detected (Xi et al. 2021).

The optimal pH and temperature of BhrPETase were determined using 4-nitrophenyl octanoate (pNO) as substrate. The pH optimum of BhrPETase was found to be in the range between 6 and 8. A drastic loss of activity was observed at pH < 6 and pH > 9. Temperature profiling of BhrPETase showed an almost linear increase from 30 °C to 90 °C (Xi et al. 2021).

The thermostability study of BhrPETase was also conducted with pNO as substrate. It was shown that BhrPETase can retain 80 % of its activity when incubated at 80 °C for 2 h. A complete loss of activity was observed when BhrPETase was incubated at 90 °C. Notably, the thermostability of BhrPETase could be improved by the addition of CaCl2, which resulted in 80% residual activity after incubation at 90 °C (Xi et al. 2021).


More information related to this part can be found in the following publications and databases:


References

Chen X., Zaro J. L., Shen, W. C. (2013): Fusion protein linkers: property, design and functionality. Advanced drug delivery reviews 65(10), 1357-1369. https://doi.org/10.1016/j.addr.2012.09.039

Kato S., Sakai S., Hirai M., Tasumi E., Nishizawa M., Suzuki K., Takai K. (2018): Long-term cultivation and metagenomics reveal ecophysiology of previously uncultivated thermophiles involved in biogeochemical nitrogen cycle. Microbes and environments 33(1), 107-110. https://doi.org/10.1264/jsme2.ME17165

Xi X., Ni K., Hao H., Shang Y., Zhao B., Qian Z. (2021): Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29. Enzyme and microbial technology 143, 109715. https://doi.org/10.1016/j.enzmictec.2020.109715


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