Part:BBa_K5117005

AtCelO

This part contains the celO gene of Acetivibrio thermocellus (synonym Clostridium thermocellum) including its native signal peptide for secretion, encoding an exoglucanase (EC 3.2.1.176).

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

Biosafety level: S1

Target organism: Bacillus subtilis

Main purpose of use: Expression in the host B. subtilis

Potential application: Degradation of cellulose

Design

For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, XbaI, SpeI, PstI and NotI were removed from the coding sequence. 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).

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1077
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Enzyme characterization according to literature

In the study of Zverlov et al. titled “A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose”, the structure of the celO gene from Clostridium thermocellum F7 is reported and the corresponding protein was shown to possess cellobiohydrolase activity (Zverlov et al. 2002).

Two truncated proteins were constructed and examined: rCelO, with the leader peptide and the dockerin module deleted (587 aa, 67.3 kDa), and rCelO-Cat, representing only the catalytic domain of CelO (415 aa, 47.9 kDa). The resulting enzymes were recombinantly produced in Escherichia coli and purified via 6x-His tag purification method. Using barley β-glucan as substrate, the optimal temperature and pH were determined to be 65 °C and 6.6, respectively (Zverlov et al. 2002).

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

• Ahmad S., Sajjad M., Altayb H. N., Sarim Imam S., Alshehri S., Ghoneim M. M., Shahid S., Mirza M. U., Nadeem M. S., Kazmi I., Waheed Akhtar, M. (2023): Engineering processive cellulase of Clostridium thermocellum to divulge the role of the carbohydrate‐binding module. Biotechnology and Applied Biochemistry 70(1), 290-305. https://doi.org/10.1002/bab.2352
• Gene sequence: https://www.ncbi.nlm.nih.gov/nuccore/AJ275975
• Protein sequence: https://www.ncbi.nlm.nih.gov/protein/CAB76938
• UniProtKB: https://www.uniprot.org/uniprotkb/Q9L3J2/entry

References

Zverlov V. V., Velikodvorskaya G. A., Schwarz W. H. (2002): A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose. Microbiology 148(1), 247-255. https://doi.org/10.1099/00221287-148-1-247