Parts Overview

Introduction

Over the summer we have worked on designing 22 parts. These parts are a selection of Polyethylene Terephthalate hydrolases (PETases), Mono(2-hydroxyethyl) Terephthate hydrolases (MHETases) and Bis(2-hydroxyethyl) Terephthalate hydrolases (BHETases). We chose these enzymes as when they work together they completely break down Polyethylene Terephthalate (PET) to the benign molecules Ethylene Glycol (EG) and Terephthalic Acid (TPA). As you may notice below, all of our BHETases are mutations of MHETase - this is due to Mono(2-hydroxyethyl) Terephthalate (MHET) and Bis(2-hydroxyethyl) Terephthalate (BHET) having very similar structures.

Our PETases

For our PETases, we used a series of mutations to try to improve their activity and thermostability. As you can see below there are common residues that are mutated. The latter four sequences include the lamB signal peptide for extracellular excretion of the enzymes.

BBa_K3039000: PETase R280A
BBa_K3039001: PETase T88A_S121E_D186H_R280A
BBa_K3039002: PETase T88A_S93M_S121E_W159F_D186H_R280A
BBa_K3039003: PETase S121E_D186H_R280A
BBa_K3039007: SP_lamB-PETase
BBa_K3039008: SP_lamB-PETase R280A
BBa_K3039009: SP_lamB-PETase W159H_S238F
BBa_K3039010: SP_lamB-PETase T88A_S93M_W159H_D186H_R280A

Our MHETases

For our MHETases we designed sequences for both wild type and a single amino acid mutation that has been shown to increase activity. MHETase is normally an intercellular enzyme. For extracellular expression we added a malE signal peptide and for periplasmic targeting we added an ompA signal peptide.

BBa_K3039004: MHETase W397A
BBa_K3039011: SP-malE-MHETase
BBa_K3039012: SP_malE-MHETase W397A
BBa_K3039014: SP_ompA-MHETase
BBa_K3039015: SP_ompA-MHETase W397A

Our BHETases

BHET is sometimes the result of the breakdown of PET using PETase. In nature the wildtype MHETase cannot normally break down BHET, however the following amino acid substitution mutations allow the MHETase to break down BHET and act as a BHETase. This allows our system to fully break down PET to ethylene glycol and terephthalic acid. For extracellular expression we added a malE signal peptide and for periplasmic targeting we added an ompA signal peptide.

BBa_K3039005: MHETase S416A_F424N
BBa_K3039006: MHETase R411A_S419G_F424N
BBa_K3039013: SP_malE-MHETase S416A_F424N
BBa_K3039016: SP_ompA-MHETase S416A_F424N

Our Ancestral Reconstructions

As part of our project, we looked at modelling ancestral mutants of PETase, the following parts are the ancestral reconstructions of this enzyme that we used.