Part:BBa_K3039012

SP_malE-MHETase W397A

Usage and Biology

The enzymes PETase and MHETase were first discovered in Ideonella sakaiensis in 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles.

This sequence is the Escherichia coli K12 (E. coli K12) codon optimized DNA of the W397A mutant MHETase with the malE signal peptide and N-terminal His-tag. The malE signal peptide has been used for extracellular production of the enzyme when modified E.coli is added to the filter system and the His-tag is attached to more easily identify the enzymes. These mutations have been reported in the literature to increase the activity of MHETase (Gottfried et al 2019).

The native predicted signal peptide (Met1-Ala19) was removed from the WT MHETase sequence (Palm et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The 27 AA malE signal peptide, which allows for excretion of the enzyme via the Sec-dependent translocation pathway (Seo et al 2019) was added to the N-terminal followed by a 13 AA His-tag. The entire amino acid sequence was codon optimised for E. coli by IDT’s on-line Codon Optimisation tool ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for TypeIIS assembly. The iGEM TypeIIS prefix and suffix were added and DNA was synthesised by IDT as a double stranded g-block. TypeIIS assembly was used to clone the resulting CDS with the T7-promoter and B0015 terminator into a high-copy number, ampicillin vector, pX1800 (University of Exeter).

Characterisation

In order to characterise our part and determine the rate of its activity and prove its functionality we have run a series of experiments. After transforming the Arctic Express, Rosetta Gami and BL21 DE3 strains of E. coli with our plasmid we induced the expression of the enzymes using IPTG. In order to confirm that the enzyme expression has been successful we ran a western blot which showed the presence of the enzyme in the soluble fractions of the sonicated cells. Afterwards the enzyme was purified and used in assays to show its functionality and determine the rate of its activity.

Western blot of the media in which the Arctic Express strain grew showing expression of extracellular MHETases. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 8. This part (SP_malE-MHETase) is labeled with 7. A clear band is visible with a size of about 65 kDa which is the size of MHETase with the His tag attached to it.

Conclusion

The enzyme is over expressed in the E. coli strain and is found in the media of the culture. Thos shows that the signal peptide has been functional and the MHETase has been secreted outside the cells. The extracellular production of MHETase could have a significant impact on future application such as possibly the scaling up of our system to the fashion mills.

References

[1] Hogyun Seo, Seongmin Kim, Hyeoncheol Francis Son, Hye-Young Sagong, Seongjoon Joo, Kyung-Jin Kim; Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli (2019) Biochem. Biophys. Res. Commun. 508(1), 250-255

[2] Gottfried J. Palm, Lukas Reisky, Dominique Böttcher, Henrik Müller, Emil A. P. Michels, Miriam C. Walczak, Leona Berndt, Manfred S. Weiss, Uwe T. Bornscheuer & Gert Weber; Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate (2019) Nat. Commun. 10(1717)

Sequences and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 615
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]