Part:BBa_K4275006

TrEGIII-t

T. reesei Endoglucanase III fused with type I dockerin can interact with type I cohesin and bind onto CipA scaffoldin of a cellulosome to achieve high-efficiency synergetic dehydrolysation of cellulose with exoglucanases and beta-glugosidases. EG randomly hydrolyzes internal amorphous regions of cellulose fibers, releasing oligosaccharides of various degrees of polymerization (DP) and consequently generating new chain ends, one reducing and one nonreducing[1].

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

Usage and Biology

TrEGIII from Glycoside hydrolases (GH)7 family cleave O-glycosidic bonds GH-catalyzed hydrolysis proceeds via a general acid mechanism involving a cyclic oxocarbenium-like transition state with protonation of the glycosidic oxygen[1]. They utilise retaining mechanisms (carboxylate substitutes glycosidic bond, then neutralised by carboxylic acid, then water attacks the ester intermediate) and randomly cleave glycosidic linkages in disordered regions of cellulose as they have relatively open active site clefts[1].

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
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 322
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 194
  

References

1. Chang, Jui-Jen et al. "PGASO: A Synthetic Biology Tool For Engineering A Cellulolytic Yeast". Biotechnology For Biofuels, vol 5, no. 1, 2012. Springer Science And Business Media LLC, https://doi.org/10.1186/1754-6834-5-53.