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Background

The strain section of our project focuses on the degradation of ethylene glycol, a chemical that is metabolized to oxalic acid further downstream. Oxalic acid is toxic to the kidney and fatal to the organism. We found an E. coli mutant from the University of Barcelona in Barcelona, Spain, that is able to grow solely on ethylene glycol, one of the two products created during PET degradation [1]. The scientists in Barcelona created these mutants through directed evolution – a process that selects for the most fit in a group under increasing conditions of ethylene glycol. From this, they learned that the main contributors in the degradation were propanediol oxidoreductase and glycolaldehyde dehydrogenase. These two enzymes are expressed at low levels in MG1655 but not at all in DH5α. We want to overexpress these enzymes in MG1655 through directed strain engineering. Rather than take the enzymes from the Barcelona strain, we want to clone the enzymes from the MG1655 and be able to control them ourselves, also known as rational strain engineering.

Rationale

Ethylene glycol is a potential toxin to any mammals that consume it, so we are taking extra measures to ensure the proper disposal of it. We want to break down the ethylene glycol product from the cutinase-driven degradation of polyethylene terephthalate (PET) so that none of the ethylene glycol is released into the environment. We have engineered our E. coli to degrade the ethylene glycol after breaking down PET so that no human intervention is necessary.
Our main goal is for the E. coli to be able to live off PET as the sole carbon source. In order to do this, it must be able to sequester the carbon into its metabolism. In the diagram below, the ethylene glycol binds to the glycolaldehyde reductase to form glycolaldehyde. After, the glycolaldehyde attaches to the glycolaldehyde dehydrogenase to form glycolate. The glycolate goes in to the metabolism via further reactions with glycolate dehydrogenase and malate synthase. The (S)-malate is the final product that is incorporated in to the citric acid (TCA) cycle. As the citric acid cycle propagates, more energy is made for the cell, allowing growth and self-sufficient development on PET.

Our Strain

Our goal in the construction of the reductase and dehydrogenase assembly is to allow a modular system for simplified testing and use. In addition to the use of the modular system, the sequencing of the strain from Barcelona shows us the other mutations in the chromosome that allow it to be efficient. Putting these two pieces of information together specifies the region in the MG1655 chromosome that we want to overexpress or mutate for efficient degradation of ethylene glycol.

What we're doing

We previously learned that the strain we had received from Barcelona possessed the ability to decompose ethylene glycol to glycolate via the enzymes glycolaldehyde reductase and glycolaldehyde dehydrogenase. Our goal was to reproduce this ability with plasmids expressed in DH5α and MG1655, two ordinary E. coli strains that cannot degrade ethylene glycol. We devised two approaches to achieve this design using psb1A3. Our first procedure involves a polycistronic system, with two genes under the control of one promoter. We will have two variants of the plasmid, one with an inducible pBAD promoter and one with the constitutive J23101 promoter.

Our second approach separates the genes, allowing us to see if the genes can be expressed more efficiently when they are under the control of one promoter each. The separation also permits us to induce one promoter and therefore express one gene at a time. With the genes expressed independently, we are able to control the production of each enzyme and ensure equal amounts are expressed. The glycolaldehyde reductase enzyme will be under the control of the pBAD promoter; the glycolaldehyde dehydrogenase enzyme will be under the control of the pLAC promoter. Because we are employing the lac promoter, we must have the lacI operon to act as the repressor. The diagrams below depict the cassette orientation within each plasmid. For each of these set-ups, we will use restriction enzymes, gel purifications, and then ligations to piece together each sub-construct. The process is lengthy in time because of the time involved for transformations, liquid cultures, and enzymatic digests.

References

1. Boronat, Albert, Caballero, Estrella, and Juan Aguilar. “Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coli.” Journal of Bacteriology, Vol. 153 No. 1, pp. 134-139, January 1983.

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