Part:BBa_K1921022

PETase+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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage

As a cell wall protein, GCW61 is often used as an anchor protein in Pichia pastoris to display some protein on the surface. By fusing GCW61 with the PETase, it can be displayed on the outer of the yeast cell wall.
By expressing the fusion protein , PETase ,which can degrade macromolecular polymers into monomerswas, expressed on the surface of Pichia pastoris,. And the whole cell catalyst for the degradation of PET was obtained. Have the PETase fixed on the cell wall, on the one hand can improve the stability of PETase, on the other hand, it is easy to control the degradation reaction of PET and PETase recycling.

Biology

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.
PETase was found from a kind of microorganism living on PET as the main carbon source. It can degrade macromolecular polymers into monomers. Surface display can reveal the protein whose gene code is coalescing the gene code of target protein or polypeptide with the counterpart of ankyrin on the surface of the host cell wall to harvest the whole cell catalyst.

Reference

[1] Kinoshita T, Fujiata M. Overview of GPI biosynthesis [J]. The enzymes. 2009;26:1-30.
[2] Orlean P, Mennon AK. Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids [J]. Journal of lipid research.2007;48(5):993-1011.
[3] Mouyna I, Fontaine T, Vai M, et al. Glycosylphosphatidy linositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall[J]. Journal of Biological Chemistry.2000;275(20):14882-14889.
[4]Shosuke Yoshida, Kazumi Hiraga, Toshihiko Takehana, Ikuo Taniguchi,Hironao Yamaji, Yasuhito Maeda, Kiyotsuna Toyohara,Kenji Miyamoto, Yoshiharu Kimura, Kohei Oda. A bacterium that degrades and assimilates poly(ethylene terephthalate) [J].science，2016（351）：1196-1199.
[5] DongHeng Guo, YanShan Xu, YaJun Kang et al (2016). Synthesis of octyl-β- d -glucopyranoside catalyzed by Thai rosewood β-glucosidase - displaying Pichia pastoris, in an aqueous/organic two-phase system[J]. Enzyme & Microbial Technology, 2016, 85:90–97.

Surface display HPLC Results

Surface display in Pichia Pastoris:

Figure 1. The activity of P. pastoris PETase-GCW61. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.

Co-display in Pichia Pastoris:

Figure 2. The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 3. The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 4. The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 5. The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 6. The activity of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformant and ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformant in best condition.