Team:RHIT/Design
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Our Design
For our project, we have designed a plasmid that secretes MHETase and the double mutant PETase to increase the rate at which PET is degraded compared to the previous PETase sequence. We inserted the plasmid into an E. coli MG1655 strain. Because of the toxicity of ethylene glycol, a second plasmid was designed to allow the bacteria to break down the ethylene glycol and utilize its products as a carbon source. These enzymes include glycolaldehyde reductase, glycolaldehyde dehydrogenase, glycolate oxidase, and malate synthase. This series of enzymes will turn the ethylene glycol, released from the breakdown of PET, into malate which can be used by the cell as a carbon source via the citric acid cycle.
Our E. coli will degrade PET into terephthalic acid (TPA) and ethylene glycol (EG) utilizing the PETase and MHETase enzymes, as shown in Figure 1 below.
Terephthalic acid is recyclable when purified and is relatively nontoxic. Ethylene glycol, on the other hand, is toxic when ingested by humans, although it has been proven to be safe for E. coli cells.
The E. coli will then take the ethylene glycol and utilize it as a carbon source using glycolaldehyde reductase, glycolaldehyde dehydrogenase, glycolate oxidase, and malate synthase. This will result in intermediates of glycolaldehyde, glycolate, and glyoxylate, as well as the product of malate which will be used in the citric acid cycle, as shown in Figure 2 below.
Two plasmids were designed to accomplish our goals. The first plasmid (Figure 3) contains PETase and MHETase, as well as the pelB secretion tag. The second plasmid (Figure 4) contains the enzyme series for the breakdown of ethylene glycol.
Our team selected the W159H/S238F double mutated enzyme based on the article released in April of 2018. The article analyzed the structure of PETase to design the specific double amino acid mutation. PETase was found to have similar common features to cutinases and lipases, which are also shown to partially degrade PET. The double mutation changes the PETase structure by narrowing the binding cleft to better resemble cutinase. In Figure 5 below, which comes from the article, E shows the proposed structure of the binding site in wild-type PETase, while F shows the proposed structure of the binding site in the W159H/S238F double mutated PETase [1].
References
- [1] Austin, H., Allen, M., Donohoe, B., Rorrer, N., Kearns, F., Silveira, R., Pollard, B., Dominick, G., Duman, R., El Omari, K., Mykhaylyk, V., Wagner, A., Michener, W., Amore, A., Skaf, M., Crowley, M., Thorne, A., Johnson, C., Woodcock, H., McGeehan, J. and Beckham, G. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19), pp.E4350-E4357.