Difference between revisions of "Part:BBa K4275039"
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===Characterization===
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==Characterization==
  
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<b>Cellulases and cellulase boosters expression</b>
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<h3>Cellulases and cellulase boosters expression</h3>
  
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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).
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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 <i>Kluyveromyces marxianus</i> with the unique origin of replication and antibiotic selection marker for the culturing of <i>Kluyveromyces marxianus</i>. 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 <i>Kluyveromyces marxianus</i> 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).
  
  
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[[Image:GreatBay SCIE--Part Fig8.png|thumbnail|750px|center|'''Figure 2:'''  
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[[Image:GreatBay SCIE--Part Fig8.png|thumbnail|750px|center|'''Figure 1:'''  
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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. ]]
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Fig.1 Construction of expression vectors for fusion proteins production in yeast <i>Kluyveromyces marxianus</i> and the analysis of the secreted enzymes (A) The design of our expression vector for production of cellulases and cellulase boosters in <i>Kluyveromyces marxianus</i>; the coding sequences for the cellulases and cellulase boosters were ligated with an alpha-mating factor secretion signal for <i>Kluyveromyces marxianus</i> 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. ]]
  
  
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<b>Cellulosome construction</b>
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<h3>Cellulosome construction</h3>
  
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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.
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We assembled the cellulose-like complex on the surface of <i>E.coli</i> by adding primary scaffold proteins, cellulases and cellulase boosters onto <i>E.coli</i> 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.
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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).
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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.
 
The overall success in engineering our project was verified by the successful construction of cellulosome complex and degrading cellulose to reducing sugars.
  
  
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[[Image:GreatBay SCIE--Part Fig9.png|thumbnail|750px|center|'''Figure 3:'''  
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[[Image:GreatBay SCIE--Part Fig9.png|thumbnail|750px|center|'''Figure 2:'''  
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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. ]]
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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. ]]
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===Sequence and Features===
 
===Sequence and Features===

Revision as of 12:50, 13 October 2022


pLAC4-KmarxMFα-MtCDH-t-tTDH1

The composite part is constructed from Kluyveromyces marxianus mating factor alpha (BBa_K4275000)[2] and cellulase booster MtCDH-t fused with type I dockerin (BBa_K4275005)[3].

The fusion of Kluyveromyces marxianus mating factor alpha (BBa_K4275000) ensures effective secretion of MtCDH-t protein by the host yeast, thus avoiding lysing the yeast cell to extract the designated cellulase booster.

Usage and Biology

Kluyveromyces marxianus mating factor alpha is an secretion signal in Kluyveromyces marxianus, which encodes for an alpha mating factor domain fused with target protein MtCDH-t. The signal peptide in alpha mating factor domain would guide the protein into RER and Golgi apparatus for the secretion of MtCDH-t.

MtCDH-t is considered a cellulase booster with strong reductive nature, enabling it to act as an electron donor to another cellulase booster TaLPMO-t during crystalline cellulose degradation. The free cellulase booster is converted into cellulosomal mode via the fusion of a type I dockerin to its C terminal. Functioning synergistically with four other cellulase and booster enzymes, MtCDH-t contributes towards effective biodegradation of cellulose components.


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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 331
    Illegal BglII site found at 3987
    Illegal XhoI site found at 1252
    Illegal XhoI site found at 4150
  • 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 2788
    Illegal AgeI site found at 1639
    Illegal AgeI site found at 2026
    Illegal AgeI site found at 2152
    Illegal AgeI site found at 2701
  • 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. 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.
4. "Part:Bba K2753052 - Parts.Igem.Org". Parts.Igem.Org, 2022, https://parts.igem.org/Part:BBa_K2753052.