Difference between revisions of "Part:BBa K3629005"

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===Usage and Biology===
 
===Usage and Biology===
  
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<i>Yarrowia lipolytica</i> is an emerging chassis in the molecular biology community. Its unique metabolic properties and efficient protein production and secretion mechanisms make it a desirable chassis for heterologous protein expression/secretion. In fact, it has been shown to have better secretory mechanisms than <i>Saccharomyces cerevisiae</i> (1). Therefore, using these chassis to secrete cellulase enzymes- which are enzymes that require high levels of secretion, is well suited.
+
<i>Yarrowia lipolytica</i> is an emerging chassis in the molecular biology community. Its unique metabolic properties and efficient protein production and secretion mechanisms make it a desirable chassis for heterologous protein expression/secretion. In fact, it has been shown to have better secretory mechanisms than <i>Saccharomyces cerevisiae</i> (1). Therefore, using this chassis to secrete cellulase enzymes- which are enzymes that require high levels of secretion, is well suited.
  
 
Fully functional cellulase is composed of:  
 
Fully functional cellulase is composed of:  
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<li>Endoglucanases (EG) which randomly cleave internal beta-bonds of cellulose polymers to make them shorter </li>
 
<li>Endoglucanases (EG) which randomly cleave internal beta-bonds of cellulose polymers to make them shorter </li>
 
<li>Cellobiohydrolases (CBH or exoglucanases) which cleave the shorter polymers to make cellobiose </li>
 
<li>Cellobiohydrolases (CBH or exoglucanases) which cleave the shorter polymers to make cellobiose </li>
 +
<ul>
 
<li>CBHI= Acts on reducing end of sugar molecule </li>
 
<li>CBHI= Acts on reducing end of sugar molecule </li>
 
<li>CBHII= Acts on non-reducing end of sugar molecule </li>
 
<li>CBHII= Acts on non-reducing end of sugar molecule </li>
 +
</ul>
 
<li>Beta-glucosidases (BGS) which cleave the cellobiose disaccharide to free glucose units </li>
 
<li>Beta-glucosidases (BGS) which cleave the cellobiose disaccharide to free glucose units </li>
 
</ol>
 
</ol>
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===Design===
 
===Design===
 +
[[Image:T--Calgary--penny.png|250px|thumb|right|Figure 1. Homology model of modified PfCBHI]]
 +
 
The native signal peptide from <i>P. funiculosum</i> was removed so it would not interfere with fused secretion tags native to <i>Y. lipolytica.</i>
 
The native signal peptide from <i>P. funiculosum</i> was removed so it would not interfere with fused secretion tags native to <i>Y. lipolytica.</i>
 +
 +
<b> CHIMERIC PROTEIN CREATION AND MODELLING </b><br>
 +
 +
To develop this modified PfCBHI we utilized a hybridized catalytic domain combining the sequence from <i>P. funiculosum</i> and <i>T. reesei</i>. These modifications allow the protein to operate at more moderate conditions for <i> Y. lipolytica </i> than the wild type protein. The linker sequence and cellulose-binding module were then added from the <i>P. funiculosum </i> wild type. These modifications were shown to increase the productivity of the protein and widen the operating parameters for the protein.
 +
 +
Structural models were generated to get a starting point for the protein. The predicted structure was then protonated in silico at numerous pHs. After protonation, the structures were solvated in water and underwent molecular dynamic simulation. Metrics around the simulations were taken and [https://2020.igem.org/Team:Calgary/Cellobiohydrolase our modelling] showed protein stability within the pH 3 to 7 range.
 +
 +
 +
<b> USAGE IN EXPRESSION CONSTRUCTS </b><br>
 +
 +
This coding sequence was attached to the Lip2 signal peptide [https://parts.igem.org/Part:BBa_K1592000 (BBa_K1592000)], the TEFin promoter [https://parts.igem.org/Part:BBa_K3629001 (BBa_K3629001)], and the XRP2 terminator [https://parts.igem.org/Part:BBa_K3629004 (BBa_K3629004)] in creation of the expression construct for this part [https://parts.igem.org/Part:BBa_K3629013 (BBa_K3629013).] We provided the fully functional expression construct in [https://2020.igem.org/Team:Calgary/Parts our collection] for teams who want to transform and use this protein directly in <i>Y. lipolytica</i>, however just the coding sequence is provided here in case teams want to use different promoters and/or signal peptides.
 +
 +
The expression constructs in our collection that can be assembled together to form a <i>Y. lipolytica</i> strain(s) that can fully degrade cellulose are:
 +
 +
<ul>
 +
<li>[https://parts.igem.org/Part:BBa_K3629012 BBa_K3629012]= <i>T. reesei</i> CBHII expression construct </li>
 +
<li>[https://parts.igem.org/Part:BBa_K3629013 BBa_K3629013]= <i>Modified P. funiculosum</i> CBHI expression construct </li>
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<li>[https://parts.igem.org/Part:BBa_K3629014 BBa_K3629014]= <i>N. crassa</i> CBHI expression construct </li>
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<li>[https://parts.igem.org/Part:BBa_K3629016 BBa_K3629016]= <i>Modified T. reesei</i> EGI expression construct </li>
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<li>[https://parts.igem.org/Part:BBa_K3629017 BBa_K3629017]= <i>T. reesei</i> EGII expression construct </li>
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<li>[https://parts.igem.org/Part:BBa_K3629018 BBa_K3629018]= <i>N. patriciarum</i> BGS expression construct </li>
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</ul>
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 +
 +
A 6x HIS affinity tag was included in this part, however not for the purpose of purification in our project, but for use in ELISA and western blot detection by using antibodies specific to the tag. This presents a cheaper and more accessible option rather than acquiring an antibody specific to the entire protein. However, future teams may choose to use this tag in purification which may be necessary in further characterization experiments. A spacer with a thrombin cleavage site was included in case the tag interferes with the protein function. Furthermore, in case this protein is expressed in tandem with the other two CBHs provided in our collection, we made all three CBHs have different affinity tags so they can be individually purified and detected as they have similar molecular weights.
 +
 +
<ol>
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<li>[https://parts.igem.org/Part:BBa_K3629005 BBa_K3629005]= Modified <i>P. funiculosum</i> CBHI with 6x His tag
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<li>[https://parts.igem.org/Part:BBa_K3629006 BBa_K3629006]= <i>N. crassa</i> CBHI with Myc tag
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<li>[https://parts.igem.org/Part:BBa_K3629007 BBa_K3629007]= <i>T. reesei</i> CBHII with FLAG tag
 +
</ol>
  
 
===Sequence and Features===
 
===Sequence and Features===
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===References===
 
===References===
  
−
1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959983/  
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1. Celińska, E., Borkowska, M., Białas, W., Korpys, P., & Nicaud, J. M. (2018). Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags. Applied microbiology and biotechnology, 102(12), 5221–5233. https://doi.org/10.1007/s00253-018-8966-9
  
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2. https://www.nature.com/articles/s41467-018-03501-8_
+
2. Taylor, L., Knott, B., Baker, J., Alahuhta, P., Hobdey, S., Linger, J., . . . Beckham, G. (2018, March 22). Engineering enhanced cellobiohydrolase activity. Retrieved October 28, 2020, from https://www.nature.com/articles/s41467-018-03501-8_
  
  

Latest revision as of 01:34, 28 October 2020


Modified Penicillium funiculosum CBHI with 6X His tag

Temperature and pH optimized cellobiohydrolase I coding sequence from Penicillium funiculosum with 6x HIS tag.

Usage and Biology

Yarrowia lipolytica is an emerging chassis in the molecular biology community. Its unique metabolic properties and efficient protein production and secretion mechanisms make it a desirable chassis for heterologous protein expression/secretion. In fact, it has been shown to have better secretory mechanisms than Saccharomyces cerevisiae (1). Therefore, using this chassis to secrete cellulase enzymes- which are enzymes that require high levels of secretion, is well suited.

Fully functional cellulase is composed of:

  1. Endoglucanases (EG) which randomly cleave internal beta-bonds of cellulose polymers to make them shorter
  2. Cellobiohydrolases (CBH or exoglucanases) which cleave the shorter polymers to make cellobiose
    • CBHI= Acts on reducing end of sugar molecule
    • CBHII= Acts on non-reducing end of sugar molecule
  3. Beta-glucosidases (BGS) which cleave the cellobiose disaccharide to free glucose units

These proteins must be in the correct proportions to each other to efficiently degrade cellulose.

CBHs provide the bulk of cellulose degradation by directionally degrading the polymers into cellobiose disaccharide units (2) While EGs enhance the ability of CBHs to act by producing more active sites for them, CBHs can still function at the ends of long cellulose polymers. Specifically, CBHI acts on the reducing ends of sugar polymers, whereas CBHII acts on the non-reducing ends. Together, these CBHs can efficiently degrade cellulose polymers from both directions.

Design

Figure 1. Homology model of modified PfCBHI

The native signal peptide from P. funiculosum was removed so it would not interfere with fused secretion tags native to Y. lipolytica.

CHIMERIC PROTEIN CREATION AND MODELLING

To develop this modified PfCBHI we utilized a hybridized catalytic domain combining the sequence from P. funiculosum and T. reesei. These modifications allow the protein to operate at more moderate conditions for Y. lipolytica than the wild type protein. The linker sequence and cellulose-binding module were then added from the P. funiculosum wild type. These modifications were shown to increase the productivity of the protein and widen the operating parameters for the protein.

Structural models were generated to get a starting point for the protein. The predicted structure was then protonated in silico at numerous pHs. After protonation, the structures were solvated in water and underwent molecular dynamic simulation. Metrics around the simulations were taken and our modelling showed protein stability within the pH 3 to 7 range.


USAGE IN EXPRESSION CONSTRUCTS

This coding sequence was attached to the Lip2 signal peptide (BBa_K1592000), the TEFin promoter (BBa_K3629001), and the XRP2 terminator (BBa_K3629004) in creation of the expression construct for this part (BBa_K3629013). We provided the fully functional expression construct in our collection for teams who want to transform and use this protein directly in Y. lipolytica, however just the coding sequence is provided here in case teams want to use different promoters and/or signal peptides.

The expression constructs in our collection that can be assembled together to form a Y. lipolytica strain(s) that can fully degrade cellulose are:


A 6x HIS affinity tag was included in this part, however not for the purpose of purification in our project, but for use in ELISA and western blot detection by using antibodies specific to the tag. This presents a cheaper and more accessible option rather than acquiring an antibody specific to the entire protein. However, future teams may choose to use this tag in purification which may be necessary in further characterization experiments. A spacer with a thrombin cleavage site was included in case the tag interferes with the protein function. Furthermore, in case this protein is expressed in tandem with the other two CBHs provided in our collection, we made all three CBHs have different affinity tags so they can be individually purified and detected as they have similar molecular weights.

  1. BBa_K3629005= Modified P. funiculosum CBHI with 6x His tag
  2. BBa_K3629006= N. crassa CBHI with Myc tag
  3. BBa_K3629007= T. reesei CBHII with FLAG tag

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 564
    Illegal BamHI site found at 1048
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 46
  • 1000
    COMPATIBLE WITH RFC[1000]

Codon optimized for expression and function in Y. lipolytica.

References

1. Celińska, E., Borkowska, M., Białas, W., Korpys, P., & Nicaud, J. M. (2018). Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags. Applied microbiology and biotechnology, 102(12), 5221–5233. https://doi.org/10.1007/s00253-018-8966-9

2. Taylor, L., Knott, B., Baker, J., Alahuhta, P., Hobdey, S., Linger, J., . . . Beckham, G. (2018, March 22). Engineering enhanced cellobiohydrolase activity. Retrieved October 28, 2020, from https://www.nature.com/articles/s41467-018-03501-8_