Part:BBa_K3629008

Modified Trichoderma reesei EGI with 6X His tag

Temperature and pH optimized endoglucanase I coding sequence from Trichoderma reesei 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 this 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.

EGs specifically are the most expressed cellulases in Trichoderma reesei while EGI has the most efficient hydrolysis on crystalline cellulose (2) The activity of endoglucanases is important to produce more exposed ends for exoglucanase or cellobiohydrolase activity.

Design

The native signal peptide from T. reesei 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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 523
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

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

2. https://pubs.rsc.org/en/content/articlelanding/2017/ra/c6ra26508a#!divAbstract