Part:BBa_K4275011

PETase5-dockerin

PETase5-dockerin is an improved version of Super-5-mut PET hydrolase from the iGEM team TJUSLS_China (Part: BBa_K3715005). This high-efficiency, thermostable, durable super mutant consists of 11 mutation sites compared to the wild-type: S214H, I168R, W159H, S188Q, R280A, A180I, G165A, Q119Y, L117F, T140D, S121E. The improvement is implemented by fusing the original sequence design with a dockerin I domain at the C' terminal to allow its high-affinity anchorage onto the CipA scaffoldin and the rest of the polyester degradation complex. The catalytic domain of PETase5-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions.

Figure 1 The 3D structure of the protein predicted by Alphafold2.

Usage and Biology

The artificially-designed PETase5-Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of K.marxianus via ScGPI, or binds to E.coli's Cell-surface Nanobody3(Nb3)(BBa_K4275026). It is believed that the immobilization of the two enzymes (PETase5-dockerin and MHETase-t(BBa_K4275010)) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.

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 88
  Illegal NgoMIV site found at 142
  Illegal NgoMIV site found at 169
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. "Part:Bba K3715005 - Parts.Igem.Org". Parts.Igem.Org, 2022, https://parts.igem.org/Part:BBa_K3715005.