Part:BBa_K4275005

MtCDH-t

MtCDH-t, fused with a dockerin, is a cellobiose dehydrogenase, one of the five cellulose-related enzymes fixed on type I cohesin, and thus on the scaffold composed of CipA2B9C and OlpB. Its reductive nature enables it to act as an electron donor to the “cellulase booster”, TaLPMO-t, another cellulose-related enzyme on the scaffold which boosts the efficiency of crystalline cellulose degradation. By fusing a type 1 dockerin through a CBM and a 36-bp glycine-rich linker at the C terminal of MtCDH, the free fungal reductase is converted into the cellulosomal mode. Synergizing with the other four cellulose-related enzymes and cellulose binding modules, MtCDH-t is an important contributor to the enhanced efficiency of cellulose degradation[1]. This is a part in a part collection where we enable efficient degradation of cellulose in textile waste.

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

Usage and Biology

MtCDH-t, fused with a dockerin, is a cellobiose dehydrogenase that enhances cellulose degradation by coupling the oxidation of cellobiose to the reductive activation of polysaccharide monooxygenases (PMO) that catalyze the insertion of oxygen into C−H bonds adjacent to the glycosidic linkage[1]. MtCDH has a heme domain at the N-terminal and a flavin domain at the C-terminal. The flavin domain is the site of oxidation of cellobiose, and subsequently, electrons are transferred to the heme domain. The reduced heme domain reduced the PMOs. MtCDH-t is fused with a type 1 dockerin through a CBM and a 36-bp glycine-rich linker, thus can bind to type 1 cohesin of the scaffold, immobilizing the enzyme.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2706
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1507
  Illegal AgeI site found at 358
  Illegal AgeI site found at 745
  Illegal AgeI site found at 871
  Illegal AgeI site found at 1420
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Phillips, Christopher M. et al. "Cellobiose Dehydrogenase And A Copper-Dependent Polysaccharide Monooxygenase Potentiate Cellulose Degradation By Neurospora Crassa". ACS Chemical Biology, vol 6, no. 12, 2011, pp. 1399-1406. American Chemical Society (ACS), https://doi.org/10.1021/cb200351y.