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

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 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. 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.

Engineered Probiotics for the Management of Congenital Metabolic Diseases: A Systematic Review

“… Jiang et al (2023) revealed that EPs with membrane-associated and intracellular PAL decreased serum Phe levels in the PKU mouse model when the enzyme was expressed in EcN. In a similar animal study by Durrer et al (2017) , the oral administration of PAL-producing EP to the PKU mouse model significantly reduced blood Phe levels.…”

“…The included studies used EcN (n=5), Lactobacillus plantarum (n=1), and L. reuteri (n=1) as hosts and manipulated them to produce intracellular (n=6) or secretory (n=1) recombinant proteins. Jiang et al (2023) revealed that EPs with membrane-associated and intracellular PAL decreased serum Phe levels in the PKU mouse model when the enzyme was expressed in EcN. In a similar animal study by Durrer et al (2017), the oral administration of PAL-producing EP to the PKU mouse model significantly reduced blood Phe levels.…”

“…In several studies, EPs were introduced as a therapeutic approach for PKU. Jiang et al (2023) revealed that membrane-associated and intracellular PAL decreased serum Phe levels in a PKU mouse model when the enzyme was expressed in EcN. PAL catalyzes the conversion of Phe to trans-cinnamic acid (TCA).…”

“…Several studies have demonstrated that EPs can be used to treat metabolic diseases ( Shen et al, 2015 ; Isabella et al, 2018 ). EPs can help reduce hyperammonemia symptoms by aiding the intestine in processing excess ammonia and converting it into nontoxic forms ( Kurtz et al, 2019 ; Jiang et al, 2023 ). Likewise, EPs can be used to catabolize Phe, providing an alternative treatment option for PKU.…”

Engineering probioticEscherichia coliNissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I CRISPR-Cas system

“…Genetically engineered EcN has shown promising application as living therapeutic in treating colitis, colorectal cancer and metabolic disease [35][36][37][38][39][40][41] . EcN has also been used as a vehicle for delivering the type II CRISPR-Cas systems to kill pathogens in animal models 42 .…”