Part:BBa_K3629010

Neocallimastix patriciarum BGS with 6X His tag

Beta-glucosidase coding sequence from Neocallimastix patriciarum 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
• 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.

BGSs are the last enzymes required to degrade cellulose to glucose. There are multiple homologues thought to be endogenously expressed by Y. lipolytica, (2) however since this is the rate limiting step of cellulose degradation (3) we are adding another recombinant BGS to the Y. lipolytica genome to enhance function. This BGS is from buffalo rumen fungus Neocallimastix patriciarum and is found to be highly efficient at using cellobiose as a substrate with an activity of 34.5U/mg (3). This enzyme also showed weak endoglucanase activity on carboxymethyl cellulose (CMC) and other short cellulose oligos. Its optimal temperature is ~40ºC and optimal pH range is 5-6 (3).

Design

This coding sequence was attached to the Lip2 signal peptide (BBa_K1592000), the TEF1 promoter (BBa_K2117000), and the XRP2 terminator (BBa_K3629004) in creation of the expression construct for this part (BBa_K3629018). 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.

We decided to use the TEF1 promoter instead of the TEFin promoter for the BGS part as Y. lipolytica already expresses a few endogenous BGSs (2), therefore such high levels of expression are not as required as the other expression constructs in our collection where the TEFin promoter was used in (BBa_K3629012-14, BBa_K36290016, and BBa_K3629023-26).

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.

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 104
  Illegal BglII site found at 2085
  Illegal BamHI site found at 417
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 928
  
• 1000
  COMPATIBLE WITH RFC[1000]

This is a long gene that might have to be split in two parts when ordered from a DNA synthesizing company. It was also 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. Guo, Z., Duquesne, S., Bozonnet, S., Cioci, G., Nicaud, J., Marty, A., & O'Donohue, M. (2017, May 19). Conferring cellulose-degrading ability to Yarrowia lipolytica to facilitate a consolidated bioprocessing approach. Retrieved October 28, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5438512/

3. JC. Tang, T., AI. Khalil, M., T. Beffa, M., Woodward, J., K. Alef, P., C. Riou, J., . . . JA. Pérezpons, X. (1970, January 01). The structural and functional contributions of β-glucosidase-producing microbial communities to cellulose degradation in composting. Retrieved October 28, 2020, from https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-018-1045-8