Part:BBa_K3629013

Modified PfCBHI expression construct with gibson homology sequences and 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.

Figure 1. Homology model of modified PfCBHI

This expression construct can be used in the assembly of a CBH gene cassette containing Modified PfCBHI (BBa_K3629013), NcCBHI (BBa_K3629014), and TrCBHII (BBa_K3629012). This gene cassette is then intended to be transformed into Y. lipolytica to create a CBH-producing strain. This strain should then be co-cultured with two other strains with a EG or BGS gene cassette. The three strains together will be able to survive on cellulose media.

CHIMERIC PROTEIN CREATION
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.

Design

The native signal peptide from P. funiculosum was removed so it would not interfere with the fused Lip2 secretion tag native to Y. lipolytica.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 2725
  Illegal EcoRI site found at 2546
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal EcoRI site found at 2546
  Illegal NheI site found at 75
  Illegal SpeI site found at 2726
  Illegal PstI site found at 2740
  Illegal NotI site found at 7
  Illegal NotI site found at 2733
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal EcoRI site found at 2546
  Illegal BglII site found at 1322
  Illegal BamHI site found at 2631
  Illegal XhoI site found at 87
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 2726
  Illegal EcoRI site found at 2546
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1
  Illegal EcoRI site found at 2546
  Illegal XbaI site found at 16
  Illegal SpeI site found at 2726
  Illegal PstI site found at 2740
  Illegal NgoMIV site found at 804
  
• 1000
  COMPATIBLE WITH RFC[1000]

There is an EcoRI site within the XRP2 terminator making this part RFC10 incompatible. However, we added the BioBrick prefix and suffix so that the other enzymes (NotI, XbaI, SpeI, and PstI) could be used to clone this part into an iGEM plasmid or another plasmid. This part can also be cloned through RFC1000 assembly.

The promoter (BBa_K3629001) is an intronic promoter, however the wild-type sequence contains BsaI, PstI, and SpeI restriction sites in the promoter section before the intron sequence. Therefore, single-nucleotides changes that match the ones in the functional BBa_K2983050 promoter were made to remove theses sites.