User:Omackraz/sandbox

Trials for the treatment of Phenylketonuria (PKU) using Escherichia coli in mouse and primate subjects are currently in progress. Escherichia coli Nissle has been genetically engineered to express genes that encode phenylalanine-metabolizing enzymes as a response to the anoxic conditions in the gut of a mammal. E. coli Nissle are often used as a live bacterial therapy to help treat human gastrointestinal diseases. These bacteria are popular for long-term clinical use due to their safety and anti-inflammatory properties. In order to treat PKU, two different metabolic pathways that aid in phenylalanine catabolism were engineered into E. coli Nissle using two different plasmids with different origins. Electroporation was used to transform the plasmids containing the pathways. The genes encoding the pathways integrated into the bacterial chromosome. These new catabolic pathways breakdown the toxic amounts of phenylalanine in the host (mammals) and accumulated less harmful products. These products can be further broken down by the hosts' pre-existing enzymes and/or excreted safely in its' urine. One of the newly inserted pathways utilizes phenylalanine ammonia lyase (PAL) and a phenylalanine transporter to convert phenylalanine into trans-cinnamate(2). Trans-cinnamate is also known as cinnamic acid. Cinnamic acid has low toxicity and is converted into hippuric acid in mammals(1). The hippuric acid is then safely excreted in the mammals' urine (3). The other pathway utilizes L-amino acid deaminase (LAAD) to convert phenylalanine into phenylpyruvate. Phenylpyruvate is also safely excreted in urine. The E. coli Nissle, now containing the two new pathways, were then engineered to include inducible growth. This period of induced growth occurs while the bacteria is being genetically modified (proving stain activity at the time of dosing) and when the bacteria are exposed to the low oxygen environment of the gut (2). After the engineered E.coli Nissle was administered to a mouse model that was diagnosed with PKU, it was shown that the phenylalanine in the mouse’s bloodstream decreased by 38% after one hour and a further 44% reduction after two hours (2). Another clinical tested showed that when the phenylalanine intake in a healthy monkey's diet was significantly increased, the newly engineered bacteria inhibited the increase in phenylalanine in the blood serum. Although further testing will be needed before moving to human trials, the success of the newly engineered phenylalanine catabolic pathways has been observed in both mouse and primate models.


 * 1) Hoskins, J. A., Holliday, S. B., & Greenway, A. M. (1984). The metabolism of cinnamic acid by healthy and phenylketonuric adults: a kinetic study. Biomedical mass spectrometry, 11(6), 296-300.
 * 2) Isabella, V. M., Ha, B. N., Castillo, M. J., Lubkowicz, D. J., Rowe, S. E., Millet, Y. A., . . . Falb, D. (2018). Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nature Biotechnology, 36(9), 857-864. doi:10.1038/nbt.4222
 * 3) National Center for Biotechnology Information. PubChem Database. Hippuric acid, CID=464, https://pubchem.ncbi.nlm.nih.gov/compound/464 (accessed on Apr. 15, 2019)