Part:BBa_K4275004

CBHII-t

CBHII-t is a fusion protein with type I dokerin (DocT) fused to the enzyme’s C terminal with a linker used between the enzyme’s catalytic domain and the dockerin module, enabling the cellulase to bind on Cipa2B9C scaffold protein by type I dockerin-cohesin interaction. CBHII-t can thus be assembled into the cellulosome complex and utilize enzyme synergy to work with endoglucanase TrEGIII-t and beta-glucosidase NpaBGS-t and accomplish a highly effective degradation of cellulose fiber in natural textiles such as cotton.

CBHII is a type of exoglucanase (cellobiohydrolase), its main function in cellulose degradation is to progressively cleave approximately two to four cellobiose units from the ends of cellulose chains by hydrolyzing the beta-1,4-glucosidic bonds. CBHII functions collectively with endoglucanase that cleaves cellulose chain internally, enabling soluble cellobiose to be released from insoluble cellulose chain, which is further processed by beta-glucosidase into glucose.

Figure 1 The 3D structure of the protein predicted by Alphafold2.

Usage and Biology

CBH or cellobiohydrolases are fungal cellulase first identified in Trichoderma reesei[1]. CBH is further categorized into CBHI and CBHII. The former cleaves the reducing ends of cellulose chain while the latter is specific towards non-reducing terminals. The activity of CBHI and CBHII is associated with GH7 and GH6 families respectively. CBHII possesses a cellulose binding domain which has a tunnel-like shape. Once the cellulose molecule enters, the hydrolyzing site of the enzyme containing a carboxylic acid and carboxylate would hydrolyze the cellulose molecule either by retaining or inverting mechanism1[1]. In retaining mechanism, the glycosidic bond undergo nucleophilic substitution by carboxylate, which is then neutralized by carboxylic acid before the water activated by carboxylate attacks the ester intermediate. In inverting mechanism, a water molecule directly dissociates the glycosidic bond.

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.2A).The successful production and secretion of the protein NpaBGS, MtCDH and TrEGIII are examined by SDS-PAGE and western blot analysis (Fig.2D).

Figure 2: Fig.2 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.3A and 3B). The overall success in engineering our project was verified by the successful construction of cellulosome complex and degrading cellulose to reducing sugars.

Figure 3: Fig.3 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
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 535
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 813
  Illegal AgeI site found at 1111
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. 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.