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. 2022 Jul 28;66(1):127–136. doi: 10.1007/s11427-021-2137-3

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

Yu Jiang 1,#, Bingbing Sun 2,3,#, Fenghui Qian 3, Feng Dong 3, Chongmao Xu 3, Wuling Zhong 4, Rui Huang 4, Qiwei Zhai 4, Yu Jiang 1,✉, Sheng Yang 2,✉
PMCID: PMC9362719  PMID: 35907113

Abstract

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 PahF263S 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.

Supporting Information

The supporting information is available online at 10.1007/s11427-021-2137-3. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

Keywords: phenylalanine ammonia lyase, cell surface display, phenylketonuria, TYS8500, oral administration

Electronic supplementary material

11427_2021_2137_MOESM1_ESM.docx (41.3KB, docx)

Supplementary material, approximately 44 KB.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21825804, 31921006), the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program”, China (2018ZX09711002-019), the Shanghai Municipal Science and Technology Major Project and the National Key Research and Development Program of China (2018YFA0800603). The authors would like to acknowledge Dr. Shuming Yin and Prof. Dali Li from Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University for providing PKU animal models in the early stage of the project. We also thank Dr. Xinwen Huang from The Children’s Hospital, Zhejiang University School of Medicine for helpful discussion on the clinical potential of the project. We also thank Jieze Zhang from the Department of Chemistry, University of Southern California for language editing of the manuscript.

Footnotes

Compliance and ethics

The author(s) declare that they have no conflict of interest. Shanghai Taoyusheng Biotechology Co., Ltd. has commercial interest in the project. All animal work conformed to the regulations of the animal ethics committee and was approved by Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences for Animal Research.

Contributed equally to this work

Contributor Information

Yu Jiang, Email: yjiang@cibt.ac.cn.

Sheng Yang, Email: syang@sibs.ac.cn.

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Supplementary Materials

11427_2021_2137_MOESM1_ESM.docx (41.3KB, docx)

Supplementary material, approximately 44 KB.

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