Expression of phenylalanine ammonia lyase as an intracellularly free and extracellularly cell surface-immobilized enzyme on a gut microbe as a live biotherapeutic for phenylketonuria

• Published in: Science China. Life sciences Vol. 66; no. 1; pp. 127 - 136
• Main Authors: Jiang, Yu, Sun, Bingbing, Qian, Fenghui, Dong, Feng, Xu, Chongmao, Zhong, Wuling, Huang, Rui, Zhai, Qiwei, Yang, Sheng
• Format: Journal Article
• Language: English
• Published: Beijing Science China Press 01-01-2023; Springer Nature B.V
• Subjects: Ammonia; Animals; Biomedical and Life Sciences; Blood levels; Cell surface; Cinnamic acid; Dietary intake; Dietary Proteins; Enzymes; Enzymes, Immobilized - therapeutic use; Gastrointestinal Microbiome; Life Sciences; Metabolic engineering; Mice; Microorganisms; Morbidity; Neurotoxicity; Nutrient deficiency; Phenylalanine; Phenylalanine - metabolism; Phenylalanine - therapeutic use; Phenylalanine Ammonia-Lyase - genetics; Phenylalanine Ammonia-Lyase - metabolism; Phenylketonuria; Phenylketonurias - genetics; Phenylketonurias - metabolism; Phenylketonurias - therapy; Probiotics; Research Paper; phenylalanine ammonia lyase; oral administration; phenylketonuria; TYS8500; cell surface display
• ISSN: 1674-7305; 1869-1889
• DOI: 10.1007/s11427-021-2137-3

Phenylketonuria (PKU), a disease resulting in the disability to degrade phenylalanine (Phe) is an inborn error with a 1 in 10,000 morbidity rate on average around the world which leads to neurotoxicity. As an potential alternative to a protein-restricted diet, oral intake of engineered probiotics degrading Phe inside the body is a promising treatment, currently at clinical stage II (Isabella, et al., 2018). However, limited transmembrane transport of Phe is a bottleneck to further improvement of the probiotic’s activity. Here, we achieved simultaneous degradation of Phe both intracellularly and extracellularly by expressing genes encoding the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL) as an intracellularly free and a cell surface-immobilized enzyme in
Escherichia coli
Nissle 1917 (EcN) which overcomes the transportation problem. The metabolic engineering strategy was also combined with strengthening of Phe transportation, transportation of PAL-catalyzed
trans
-cinnamic acid and fixation of released ammonia. Administration of our final synthetic strain TYS8500 with PAL both displayed on the cell surface and expressed inside the cell to the
Pah
F263S
PKU mouse model reduced blood Phe concentration by 44.4% compared to the control EcN, independent of dietary protein intake. TYS8500 shows great potential in future applications for PKU therapy.