Part:BBa_K4275038

pLAC4-KmarxMFα-CBHII-t-tTDH1

The composite part contains a pLac4 promoter[1], tTDH1 terminator[4], Kluyveromyces marxianus mating factor α (BBa_K4275000)[2] and exoglucanase CBHII-t (cellobiohydrolase) fused with type I dockerin that constitute the assembly of cellulosome complex [3].

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.

Characterization

Cellulases and cellulase boosters expression

The enzymatic digestion of the polysaccharide chains of cellulose was completed by exoglucanase, endoglucanase and 1-4 betaglucosidase, and this series of reactions are catalysed by LPMO and CDH. We constructed expression vectors for yeast Kluyveromyces marxianus with the unique origin of replication and antibiotic selection marker for the culturing of Kluyveromyces marxianus. Expression vectors were made distinct by the insertion of different sequences coding for the ligated form of the cellulase enzymes, LPMO and CDH. The enzymes were ligated with an alpha-mating factor secretion signal for Kluyveromyces marxianus at the N-terminus and a type I dockerin domain at the C-terminus (Fig.1A).The successful production and secretion of the protein NpaBGS, MtCDH and TrEGIII are examined by SDS-PAGE and western blot analysis (Fig.1D).

Figure 1: Fig.1 Construction of expression vectors for fusion proteins production in yeast Kluyveromyces marxianus and the analysis of the secreted enzymes (A) The design of our expression vector for production of cellulases and cellulase boosters in Kluyveromyces marxianus; the coding sequences for the cellulases and cellulase boosters were ligated with an alpha-mating factor secretion signal for Kluyveromyces marxianus at the N terminus and a type I dockerin domain at the C terminus linked by a flexible linker (B) The growth curve of recombinant yeasts transformed with expression plasmids coding for different enzymes (C) The agarose gel electrophoresis image of coding sequences for different enzymes, respectively NpaBGS, TaLPMO, CBHII, MtCDH and TrEGIII (D) Western blot result for TrEGIII and MtCDH.

Cellulosome construction

We assembled the cellulose-like complex on the surface of E.coli by adding primary scaffold proteins, cellulases and cellulase boosters onto E.coli expressing secondary scaffold proteins. The mixture was centrifuged and resuspended in tris-HCl. The mixture underwent centrifugation and resuspension using tris-HCl, and cellulose was added to the mixture. After 24h, the mixture was filtered and tested for glucose by Benedict's test. From the result, we determined that the cellulosome-like complexes are able to degrade cellulose at a higher efficiency than cell-free cellulases mixture (Fig.2A and 2B). The overall success in engineering our project was verified by the successful construction of cellulosome complex and degrading cellulose to reducing sugars.

Figure 2: Fig.2 The Benedict’s quantitative and qualitative tests for reducing sugar produced by the enzymatic or cellulosomal degradation of cellulose (A) Benedict’s qualitative test result for reducing sugar production through 24h of cellulose degradation by cellulosome, cellulosome without boosters, nanobody presenting cell+free cellulases+cellulase boosters, nanobody presenting cell+cellulases and nanobody presenting cell control from left to right (B) Benedict’s quantitative test for absorbance of the samples obtained from the Benedict’s qualitative test at 635 nm wavelength.

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.