Chassis or strain engineering focuses on the modification of chromosomes instead of plasmids and encompasses both directed evolution
and rational engineering. This part of the project focuses on the elimination of ethylene glycol, a degradation product of PET that is metabolized to oxalic acid further downstream the metabolic pathway. Oxalic acid is toxic to the kidney and fatal to the organism (2). (For a look at how we handled these compounds safely, look at our Safety Page!)
Once we discovered the potential toxicity of our project, we searched high and low to find ways to eliminate ethylene glycol (EG) as much as possible. In the scientific literature, we found that no wild-type E. coli can utilize EG (1). However, we did find an E. coli mutant, called Strain E-15 EG3, from the University of Barcelona, that is able to grow on ethylene glycol alone (1). While the University of Barcelona's paper on Strain E-15 EG3 was published nearly 30 years ago, we contacted the authors and asked for the strain to perform our own testing. The researchers were able to find the strain in one of their freezers and ship it to our lab. We have dubbed the rediscovery of the strain "Freezer Archaeology".
By performing various experiments on MG1655 including directed evolution, which selects the cells with the most fitness in a population, and ethyl methylsulfonate (EMS), which introduces random mutations to a population, the scientists 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 worked to overexpress these enzymes in MG1655 through directed strain engineering, and also clone the enzymes from MG1655 for more control, by performing our own directed evolution, EMS, rational engineering, and site-directed mutagenesis experiments.
Pathway Goals
Since ethylene glycol (EG) is a potential toxin to any mammals that consume it, we worked to break EG down as efficiently as possible so that none of the ethylene glycol is released into the environment. We have engineered our E. coli to degrade ethylene glycol 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.
Note: starred enzymes are what we are using in our construct
Our Strain
We wanted to assemble reductase and dehydrogenase to allow a modular system for simplified testing and use when compared to chromosomal testing. In addition to the use of the modular system, the sequencing of Strain E-15 EG3 shows us the other mutations in the chromosome that allow it to utilize ethylene glycol. Extracting the data from sequencing and modular testing together will help us identify the region in the MG1655 chromosome that we want to overexpress or mutate for efficient degradation of ethylene glycol.
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.
2. Bsc, S. N. and Gp Savage Bsc(hons), PhD, Nz Reg NutR. (1999), Oxalate content of foods and its effect on humans. Asia Pacific Journal of Clinical Nutrition, 8: 64–74. doi: 10.1046/j.1440-6047.1999.00038.x
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