Part:BBa_K3629009

Trichoderma reesei EGII with FLAG tag

Endoglucanase II coding sequence from Trichoderma reesei with FLAG 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
• 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.

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

This coding sequence was attached to the XPR2 signal peptide (BBa_K3629000), the EXP promoter (BBa_K3629002), and the XRP2 terminator (BBa_K3629004) in creation of the expression construct for this part (BBa_K3629017). In previous studies, this expression construct produced 132mg/L of enzyme (3). We provided the fully functional expression construct in our collection for teams who want to transform and use this protein directly in Y. lipolytica, however just the coding sequence is provided here in case teams want to use different promoters and/or signal peptides.

The expression constructs in our collection that can be assembled together to form a Y. lipolytica strain(s) that can fully degrade cellulose are:

• BBa_K3629012= T. reesei CBHII expression construct
• BBa_K3629013= Modified P. funiculosum CBHI expression construct
• BBa_K3629014= N. crassa CBHI expression construct
• BBa_K3629016= Modified T. reesei EGI expression construct
• BBa_K3629017= T. reesei EGII expression construct
• BBa_K3629018= N. patriciarum BGS expression construct

A 6x HIS affinity tag was included in this part, however not for the purpose of purification in our project, but for use in ELISA and western blot detection by using antibodies specific to the tag. This presents a cheaper and more accessible option rather than acquiring an antibody specific to the entire protein. However, future teams may choose to use this tag in purification which may be necessary in further characterization experiments. A spacer with a thrombin cleavage site was included in case the tag interferes with the protein function. Furthermore, in case this protein is expressed in tandem with the other EG provided in our collection, we made both EGs have different affinity tags so they can be individually purified and detected as they have similar molecular weights.

1. BBa_K3629008= Modified T. reesei EGI with 6x His tag
2. BBa_K3629009= T. reesei EGII with FLAG tag

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 283
  Illegal NgoMIV site found at 817
  Illegal NgoMIV site found at 1135
  
• 1000
  COMPATIBLE WITH RFC[1000]

Codon optimized for expression and function in Y. lipolytica.

References

1. Celińska, E., Borkowska, M., Białas, W., Korpys, P., & Nicaud, J. (2018, June). Robust signal peptides for protein secretion in Yarrowia lipolytica: Identification and characterization of novel secretory tags. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959983/

2. Wang, X., Rong, L., Wang, M., Pan, Y., Zhao, Y., & Tao, F. (2017, September 29). Improving the activity of endoglucanase I (EGI) from Saccharomyces cerevisiae by DNA shuffling. Retrieved from https://pubs.rsc.org/en/content/articlelanding/2017/ra/c6ra26508a

3. Wei, H., Wang, W., Alper, H., Xu, Q., Knoshaug, E., Van Wychen, S., . . . Zhang, M. (2019, January 9). Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333634/