Part:BBa_K4275002

NpaBGS-t

NpaBGS-t is fused with type I dockerin to enable the cellulase to interact with type I cohesin on Cipa2B9C scaffold and displayed on cellulosome complex. This type I dockerin-cohesin interaction is an extremely strong non-covalent interaction between molecules that stabilizes the structure of cellulosome. NpaBGS-t would act collectively with two other cellulases: an endoglucanase called TrEGIII-t and an exoglucanase called CBHII-t.

Beta-glucosidase is involved in the catalyzing the hydrolytic degradation of plant polysaccharide cellulose. In cellulose degradation, endoglucanase randomly cuts the beta-1,4-glycosidic bonds along cellulose chains, producing cellulose fragments of various length[1]. Exoglucanase then acts on either reducing or non-reducing ends of cellulose to free cellobiose molecules. Beta-glucosidase functions by converting cellobiose and oligosaccharides into glucose. As cellobiose is a strong allosteric inhibitor for the activity of both endo-beta-1,4-glucanase and cellodextrinase, beta-glucosidase plays an essential role in enhancing the efficiency of cellulose degradation[1].

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

Usage and Biology

NpaBGS is cDNA coding beta-glucosidase isolated from buffalo rumen fungus Neocallimastix patriciarum W5.

NpaBGS has 776 amino acid residues with a molecular mass of 85.1 kDa. Beta-glucosidase genes are commonly placed into glycosyl hydrolase families 1 and 3 (GH1 and GH3)[1]. NpaBGS is considered a beta-glucosidase of GH3 carrying two conserved putative domain - a GH3 N-terminal domain (Pfam00933) and a GH3 C-terminal domain (Pfam01915), located at residues 62 ~270 and 350 ~ 577 respectively. The aspartic acid residue Asp-251 in the conserved domain might be the active site of NpaBGS[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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 502
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1577
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1074
  Illegal AgeI site found at 88
  Illegal AgeI site found at 106
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1.Chen, Hsin-Liang et al. "A Highly Efficient Β-Glucosidase From The Buffalo Rumen Fungus Neocallimastix Patriciarum W5". Biotechnology For Biofuels, vol 5, no. 1, 2012. Springer Science And Business Media LLC, https://doi.org/10.1186/1754-6834-5-24.