Difference between revisions of "Part:BBa K3629007"

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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 this chassis to secrete cellulase enzymes- which are enzymes that require high levels of secretion, is well suited.
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Fully functional cellulase is composed of:
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<ol>
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<li>Endoglucanases (EG) which randomly cleave internal beta-bonds of cellulose polymers to make them shorter </li>
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<li>Cellobiohydrolases (CBH or exoglucanases) which cleave the shorter polymers to make cellobiose </li>
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<li>CBHI= Acts on reducing end of sugar molecule </li>
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<li>CBHII= Acts on non-reducing end of sugar molecule </li>
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<li>Beta-glucosidases (BGS) which cleave the cellobiose disaccharide to free glucose units </li>
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</ol>
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These proteins must  be in the correct proportions to each other to efficiently degrade cellulose.
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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===
 
===Design===

Revision as of 00:06, 27 October 2020


Trichoderma reesei CBHII with FLAG tag

Cellobiohydrolase II coding sequence from Trichoderma reesei with FLAG 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
  3. CBHI= Acts on reducing end of sugar molecule
  4. CBHII= Acts on non-reducing end of sugar molecule
  5. 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

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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 341
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 601
  • 1000
    COMPATIBLE WITH RFC[1000]

References