Part:BBa_K1921025

sJanus+linker.b+GCW61

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 187
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 196
  

Usage

sJanus-GCW61 is a construction we designed, which aims to change the hydrophobicity of the outer surface of the cell wall. GCW61 is an anchoring protein derived from Pichia GS115. When fused with a certain protein on its C-terminus, such as sJanus, this protein can be displayed on to the cell wall. Thus, sJanus, a class II hydrophobin, can eventually manage to increase the hydrophobicity of the cell wall, having displayed on cell surface, and ultimately help in targeting hydrophobic substrates when the host cell is a whole cell biocatalyst. Another usage of this construct is to protect the host cell from severe or extreme environments, since hydrophobins are considered to be very stable and protective.
We co-displayed PETase-GCW21/51/61 along with sJanus-GCW61, in attempt to make our whole cell biocatalyst more efficient and tolerant. Previous studies on co-displaying hydrophobin with lipases have already shown positive results on the promotion of efficiency. It is confirmed that most lipases are more active when entering a more hydrophobic environment. Since PETase is also classified as lipase, we decided to build this construction and make this attempt.

Biology

Janus could be produced by filamentous fungi, such as Ascomycetes and Basidiomycetes, and their scientific name is hydrophobin. Many different aspects of fungal development have been attributed to Janus. For example, they are thought to play a role in the formation of aerial hyphae and fruiting bodies. One of the most important features of Janus is that they are able to assemble spontaneously into amphipathic monolayers at hydrophobic–hydrophilic interfaces.
There are two classes of Janus, which are divided by the stability of their self-assembly. sJanus from Trichoderma reesei belongs to Class II. The assemblies of class II Janus can be dissolved in ethanol or sodium dodecyl sulfate or through the application of pressure or lowering of the temperature.
GCW61 was gained from Pichia pastoris GS115.
As one of the Glycosylphosphatidylinositoled cell wall proteins (GPI-CWPs), GCW61 is located in the outer layer of yeast cell wall, its C terminal is oligo mannose glycosylated. Subsequently, the mannose chain of GCW61 connect with the β-1,6 dextranomer of inner cell wall layer by forming covalent connection, thus, the GCW61 is fixed in the outer layer of the cell wall protein.

Reference

[1] Shosuke Y, Kazumi H, Toshihiko T, Ikuo Taniguchi, Hironao Y, Yasuhito M, Kiyotsuna T, Kenji M, Yoshiharu K, Kohei O. 2016. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196-1199. DOI: 10.1126/science.aad6359.
[2] Pan W, Jie H, Yufei S, Matthew R, Li Z, Shuangyan H, Shuli L, Haixin S, Ying L. 2016. Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B. Appl Microbiol Biotechnol DOI 10.1007/s00253-016-7431-x
[3] Brzozowski A, Derewenda U, Derewenda Z, Dodson G, Lawson D, Turkenburg J, Bjorkling F, Huge-Jensen B, Patkar S, Thim L . 1991. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 351(6326):491–494. doi:10.1038/351491a0