Part:BBa_K5117006

AtCelS

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

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

Target organism: Bacillus subtilis

Main purpose of use: Gene expression and protein production using the host Bacillus subtilis

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 XhoI site found at 1471
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 943
  
• 1000
  COMPATIBLE WITH RFC[1000]

Enzyme characterization according to literature

In the study of Kruus et al. named “Exoglucanase activities of the recombinant Clostridium thermocellum CelS, a major cellulosome component”, the exoglucanase celS from Clostridium thermocellum was recombinantly produced and characterized (Kruus et al. 1995).

CelS protein was expressed in Escherichia coli and subsequently purified from the formed inclusion bodies. For that, the inclusion bodies were treated with 5 M urea first. Then, proteins were dialyzed and incubated at 60 °C for 10 min in 10 mM CaCl2. Next, ion exchange chromatography was performed. The collected fraction with CelS was dialyzed again and then used for further experiments. The purified recombinant protein had a molecular weight of 86 kDa as expected (Kruus et al. 1995).

Initially, the activity of CelS was tested with different substrates, for example with carboxymethylated cellulose, amorphous cellulose, avicel or xylan. The best performance of CelS was observed with amorphous cellulose. Furthermore, the degradation products of avicel were studied with HPLC method. The major product obtained was cellobiose, indicating that CelS is an exoglucanase (Kruus et al. 1995).

The optimal temperature and pH of CelS were determined with amorphous cellulose as substrate. The best performance of CelS was achieved at 70 °C and pH 5.7 (Kruus et al. 1995).

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

• Guimarães, B. G. et al., The Crystal Structure and Catalytic Mechanism of Cellobiohydrolase CelS, the Major Enzymatic Component of the Clostridium thermocellum cellulosome (2002) https://doi.org/10.2210/pdb1l1y/pdb
• Guimarães B. G., Souchon H., Lytle B. L., Wu J. D., Alzari P. M. (2002): The crystal structure and catalytic mechanism of cellobiohydrolase CelS, the major enzymatic component of the Clostridium thermocellum cellulosome. Journal of molecular biology 320(3), 587-596. https://doi.org/10.1016/S0022-2836(02)00497-7
• Wang W. K., Kruus K., Wu, J. H. (1993): Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component. Journal of bacteriology 175(5), 1293-1302. https://doi.org/10.1128/jb.175.5.1293-1302.1993
• Wang W. K. & Wu, J. D. (1993): Structural features of the Clostridium thermocellum cellulase Ss gene. Applied biochemistry and biotechnology 39, 149-158. https://doi.org/10.1007/BF02918985
• Wilson C. M., Rodriguez M., Johnson C. M., Martin S. L., Chu T. M., Wolfinger R. D., Hauser L. J., Land M. L., Klingeman D. M., Syed M. H., Ragauskas A. J., Tschaplinski T. J., Mielenz J. R., Brown S. D. (2013): Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass. Biotechnology for biofuels 6, 1-18. https://doi.org/10.1186/1754-6834-6-179
• Gene sequence: https://www.ncbi.nlm.nih.gov/nuccore/L06942
• Protein sequence: https://www.ncbi.nlm.nih.gov/protein/ABN53296
• UniProtKB: https://www.uniprot.org/uniprotkb/A3DH67/entry

References

Kruus K., Wang W. K., Ching J., Wu J. H. (1995): Exoglucanase activities of the recombinant Clostridium thermocellum CelS, a major cellulosome component. Journal of bacteriology 177(6), 1641-1644. https://doi.org/10.1128/jb.177.6.1641-1644.1995