Difference between revisions of "Part:BBa K3629018"
(→‎Usage and Biology)
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__TOC__
 
__TOC__
 
===Usage and Biology===
 
===Usage and Biology===
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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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<ul>
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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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</ul>
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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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BGSs are the last enzymes required to degrade cellulose to glucose. There are multiple homologues thought to be endogenously expressed by <i>Y. lipolytica</i>, (1) however since this is the rate limiting step of cellulose degradation (2) we are adding another recombinant BGS to the <i>Y. lipolytica</i> genome to enhance function. This BGS is from buffalo rumen fungus <i>Neocallimastix patriciarum</i> and is found to be highly efficient at using cellobiose as a substrate with an activity of 34.5U/mg (2). This enzyme also showed weak endoglucanase activity on carboxymethyl cellulose (CMC) and other short cellulose oligos. Its optimal temperature is ~40ºC  and optimal pH range is 5-6 (2).
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This expression construct can be used to assemble a BGS gene cassette. This gene cassette is then intended to be transformed into <i>Y. lipolytica</i> to create a BGS-producing strain. This strain should then be co-cultured with two other strains with either a CBH or EG gene cassette. The three strains together will be able to survive on cellulose media.
  
 
===Design===
 
===Design===

Revision as of 06:51, 27 October 2020


N. patriciarum BGS expression construct with 6X His tag

Usage and Biology

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.

BGSs are the last enzymes required to degrade cellulose to glucose. There are multiple homologues thought to be endogenously expressed by Y. lipolytica, (1) however since this is the rate limiting step of cellulose degradation (2) we are adding another recombinant BGS to the Y. lipolytica genome to enhance function. This BGS is from buffalo rumen fungus Neocallimastix patriciarum and is found to be highly efficient at using cellobiose as a substrate with an activity of 34.5U/mg (2). This enzyme also showed weak endoglucanase activity on carboxymethyl cellulose (CMC) and other short cellulose oligos. Its optimal temperature is ~40ºC and optimal pH range is 5-6 (2).

This expression construct can be used to assemble a BGS gene cassette. This gene cassette is then intended to be transformed into Y. lipolytica to create a BGS-producing strain. This strain should then be co-cultured with two other strains with either a CBH or EG gene cassette. The three strains together will be able to survive on cellulose media.

Design

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3112
    Illegal SpeI site found at 253
    Illegal PstI site found at 218
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3112
    Illegal NheI site found at 44
    Illegal SpeI site found at 253
    Illegal PstI site found at 218
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3112
    Illegal BglII site found at 660
    Illegal BglII site found at 2641
    Illegal BamHI site found at 973
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3112
    Illegal SpeI site found at 253
    Illegal PstI site found at 218
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3112
    Illegal SpeI site found at 253
    Illegal PstI site found at 218
    Illegal NgoMIV site found at 1484
  • 1000
    COMPATIBLE WITH RFC[1000]