Part:BBa_K1921026
inJanus+linker.b+GCW61
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 121
Usage
inJanus-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 inJanus, this protein can be displayed on to the cell wall. Thus, inJanus, a class I 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 inJanus-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. inJanus from Grifola frondosa belongs to Class I. They could generate very insoluble assemblies, which can only be dissolved in strong acids such as trifluoroacetic acid or formic acid.
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
Co-display HPLC Results
Figure 1. The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.
Figure 2. The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.
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