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 [1]. 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.

Characterization

PET degradation

The degradation of PET polymers using an enzymatic approach requires the synergetic functions of PETase and MHETase, producing terephthalic acids and ethylene glycol by hydrolytic cleavages (Fig.2A). We constructed E.coli expression vectors for the production of PETase and MHETase that can be induced by IPTG (Fig.2B). The production of the proteins was verified by SDS-PAGE analysis (Fig.2C).

Figure 2: Fig.2 FAST-PETase expression (A) Metabolic pathway of PET degradation, PETase catalyzes the cleavage of PET into MHET (mono-2-hydroxyethyl terephthalate) and EG (Ethylene glycol). (B) Genetic circuit constructions of FAST-PETase and FAST-PETase-t with type I dockerin fused to anchor the enzyme subunit onto the cellulosome complex. (C) SDS-page analysis for PETase 5 and PETase 5-t (D) The PH values of different samples of PET degraded by PETases either fused or not fused with type I dockerin domain.

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.