Coding

Part:BBa_K3629005

Designed by: Sravya Kakumanu   Group: iGEM20_Calgary   (2020-10-11)
Revision as of 23:34, 26 October 2020 by Sravyakakumanu (Talk | contribs) (→‎Usage and Biology)


Modified Penicillium funiculosum CBHI with 6X His tag

Temperature and pH optimized cellobiohydrolase I coding sequence from Penicillium funiculosum with 6x HIS 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 these 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 P. funiculosum 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 BglII site found at 564
    Illegal BamHI site found at 1048
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 46
  • 1000
    COMPATIBLE WITH RFC[1000]

Codon optimized for expression and function in Y. lipolytica.

References

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959983/


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