Part:BBa_K4275038

pLAC4-KmarxMFα-CBHII-t-tTDH1

The composite part contains a pLac4 promoter, tTDH1 terminator, Kluyveromyces marxianus mating factor α (BBa_K4275000) and exoglucanase CBHII-t (cellobiohydrolase) fused with type I dockerin that constitute the assembly of cellulosome complex.

The fusion of Kluyveromyces marxianus mating factor α enables the successful secretion of CBHII-t cellulase from the host yeast; the type I dockerin fused onto CBHII-t allows the cellulase to bind to type I cohesin on the CipA scaffoldin through cohesin-dockerin interaction and is thus assembled into the cellulosome complex that is anchored on the host.

Usage and Biology

Kluyveromyces marxianus mating factor α encodes for an α-mating factor (αMF) domain fused with CBHII-t. It functions as a secretion signal; the signal peptide expressed in the αMF domain directs the cellulase fused with it through endoplasmic reticulum, Golgi body and eventually secreted, allowing the CBHII-t to be secreted outside from the host cell, resulting in the successful integration of cellulosome complex outside the host.

CBHII-t is an exoglucanase (cellobiohydrolase) with type I dockerin (DocT) fused onto the C terminal of the enzyme with a linker used between the enzyme’s catalytic domain and the dockerin module. The CBHII enzyme possesses a cellulose binding domain and can progressively cleave cellobiose units from the ends of cellulose chains by hydrolyzing the beta-1,4-glycosidic bonds. The integration of CBHII-t into the cellulosome enables it to work in a synergy with other cellulases and significantly boost the degradation efficiency of the cellulosome complex.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 331
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 331
  Illegal NheI site found at 89
  Illegal NheI site found at 1816
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 331
  Illegal XhoI site found at 1252
  Illegal XhoI site found at 2962
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 331
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 331
  Illegal NgoMIV site found at 2094
  Illegal AgeI site found at 2392
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Rajkumar, Arun S. et al. "Biological Parts For Kluyveromyces Marxianus Synthetic Biology". Frontiers In Bioengineering And Biotechnology, vol 7, 2019. Frontiers Media SA, https://doi.org/10.3389/fbioe.2019.00097.
2.“Mating Factor Alpha-1 [Kluyveromyces Marxianus] - Protein - NCBI.” National Center for Biotechnology Information, U.S. National Library of Medicine, https://www.ncbi.nlm.nih.gov/protein/QGN17207.1
3. Chang, Jui-Jen et al. "Assembling A Cellulase Cocktail And A Cellodextrin Transporter Into A Yeast Host For CBP Ethanol Production". Biotechnology For Biofuels, vol 6, no. 1, 2013. Springer Science And Business Media LLC, https://doi.org/10.1186/1754-6834-6-19.
4. "Part:Bba K2753052 - Parts.Igem.Org". Parts.Igem.Org, 2022, https://parts.igem.org/Part:BBa_K2753052.