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 (figure 1) 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

Table 1. This table presents the restriction enzymes that can be used to digest this expression construct and expose the ends of the built-in Gibson homology sequences to T5 exonuclease. T5 exonuclease requires a blunt end or sticky end at the 5’ end to start degrading a DNA strand from 5’ to 3’.

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

GIBSON ASSEMBLY

This expression construct was designed to be assembled with other expression constructs from our collection via Gibson Assembly:

• BBa_K3629015= Nourseothricin resistance expression construct
• BBa_K3629013= Modified P. funiculosum CBHI expression construct
• BBa_K3629014= N. crassa CBHI expression construct
• BBa_K3629012= TrCBHII expression construct
• BBa_K3629016= Modified T. reesei EGI expression construct
• BBa_K3629017= T. reesei EGII expression construct

To use this part in the various Gibson assemblies, it must first be digested by a specific restriction enzyme to expose the end of a specific Gibson homology sequence for T5 exonuclease (see table 1 to see which restriction enzymes expose which Gibson homology sequences). Furthermore, this part, and any accompanying parts, must be assembled into a destination vector containing BBa_K3629015 (nourseothricin resistance expression construct) using the Gibson 1 and 2 homology sequences. Following assembly with BBa_K369015, the full gene cassette can be linearized with NotI and transformed into Y. lipolytica with the nourseothricin selection marker (figure 2). To watch a short animation on how this works click here.

The functions of the Gibson homology sequences built-in to this part are:

• Gibson 1-2= To assemble with the nourseothricin destination vector (BBa_K3629015)
• Gibson 5-8= To assemble with the other expression constructs listed above
• Gibson Y and X= To swap the TEFin promoter with the TEF1 promoter (BBa_K3629003)

Figure 2. Assembly of multiple expression constructs into one gene cassette using the nourseothricin resistance construct (BBa_K3269015) as the destination vector.

Table 2. Creation of three Yarrowia lipolytica strains each secreting one class of cellulase enzymes. The parts in the first column should be individually digested by the corresponding enzymes in the second column. All of the digested products should then be put in the same tube with the Gibson reagents to create the resulting plasmid/gene cassette in column three.

The expression constructs mentioned above were designed to be assembled together in different combinations. Over 20 different gene cassettes can be formed using these parts. The full tables outlining the assembly of these different cassettes can be found here.

To create the three strains of Y. lipolytica that contain the different cellulase gene cassettes mentioned previously, the individual expression constructs must be digested as per table 2. The digestion products can then be put together and mixed with the Gibson reagents for the final gene cassettes to form.

CONSTRUCT COMPONENTS

1. Modified PfCBHI Coding sequence (BBa_K3629005)
2. TEFin promoter (BBa_K3629001) for high expression
3. A 6x HIS affinity tag, however not for the purpose of purification, but for use in ELISA and western blot detection by using antibodies specific to the tag. This presents a cheaper and more accessible alternative to acquiring an antibody specific to the entire protein. A spacer with a thrombin cleavage site was included in case the affinity tag needed to be removed after translation
4. The Lip2 (BBa_K1592000) signal peptide sequence for secretion
5. XRP2 terminator was used for its short and compact sequence

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.