Part:BBa_K3629012

T. reesei CBHII expression construct with gibson homology sequences and FLAG 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.

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 creating 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.

Design

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

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 create the three strains of Y. lipolytica that could be co-cultured for full cellulase activity, the individual expression constructs must be digested as per table 1. The digestion products can then be put together and mixed with the Gibson reagents for the final gene cassettes to form (table 1).

Table 1. 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.

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Parts to add (DNA Part names)	Digest parts with1	Resulting plasmid (composition)
Strain 1 BBa_K3629016 (Modified TrEGI construct) BBa_K3629017 (TrEGII construct) BBa_K3629015 (nourseothricin construct)	1.    BbsI and SalI 2.    SalI and SmaI 3.    SmaI	Modified TrEGI + TrEGII + Nourseothricin2
Strain 2 BBa_K3629013 (Modified PfCBHI construct) BBa_K3629014 (NcCBHI construct) BBa_K3629012 (TrCBHII construct) BBa_K3629015 (nourseothricin construct)	1.    BbsI and SmaI 2.    NcoI and SalI 3.    SalI and NcoI 4.    SmaI	Modified PfCBHI + NcCBHI + TrCBHII + Nourseothricin
Strain 3 BBa_K3629018 (NpBGS construct) BBa_K3629015 (nourseothricin construct)	1.    SmaI and BbsI 2.    SmaI	NpBGS + Nourseothricin

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¹Digest with these enzymes to expose the appropriate Gibson homology sequence to create the desired plasmid in column three.
²Nourseothricin= Nourseothricin resistance gene

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 2737
  Illegal EcoRI site found at 2561
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal EcoRI site found at 2561
  Illegal NheI site found at 75
  Illegal SpeI site found at 2738
  Illegal PstI site found at 2752
  Illegal NotI site found at 7
  Illegal NotI site found at 2745
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal EcoRI site found at 2561
  Illegal BamHI site found at 2686
  Illegal XhoI site found at 124
  Illegal XhoI site found at 1198
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 2738
  Illegal EcoRI site found at 2561
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1
  Illegal EcoRI site found at 2561
  Illegal XbaI site found at 16
  Illegal SpeI site found at 2738
  Illegal PstI site found at 2752
  Illegal NgoMIV site found at 1458
  
• 1000
  COMPATIBLE WITH RFC[1000]