Part:BBa_K4275008
FAST-PETase-t
FAST-PETase-t is a dockerin-fused variant of free FAST-PETase (BBa_K4275007). The enzyme carries the same amino acid mutations (N233K, R224Q, S121E, D186H, and R280A)[1] as the former and shares all the common sequences in the CDS, except the C' terminal fusion of a type-I dockerin domain in this enzyme. The catalytic domain of FAST-PETase-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions. The type-I dockerin domain forms high-affinity non-covalent interaction with type-I cohesins on the cipA scaffoldin.
Figure 1 The 3D structure of the protein predicted by Alphafold2.
Usage and Biology
The artificially-designed FAST-PETase - 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 (FAST-PETase-t and MHETase-t) 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
1. Lu, Hongyuan et al. "Machine Learning-Aided Engineering Of Hydrolases For PET Depolymerization". Nature, vol 604, no. 7907, 2022, pp. 662-667. Springer Science And Business Media LLC, https://doi.org/10.1038/s41586-022-04599-z.
| None |