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bioRxiv · 10.64898/2026.03.19.713057

Substrate transport limits phenylalanine ammonia-lyase activity in engineered Lacticaseibacillus rhamnosus GG

Choudhury, D.·Mays, Z. J.·Nair, N. U.
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Abstract

Probiotic-based encapsulation offers unique advantages over purified enzymes, such as increased protection from thermal-, pH-, and protease-mediated degradation, for oral therapeutic delivery applications. However, one of the major disadvantages of whole-cell systems is lower reaction rate due to substrate-product transport limitations imposed by the cell membrane and/or wall. In this work, we explore the potential of different lactic acid bacteria (LAB) - Lacticaseibacillus rhamnosus GG (LGG), Lactococcus lactis (Ll), and Lactiplantibacillus plantarum (Lp) - as expression hosts for recombinant Anabaena variabilis phenylalanine ammonia-lyase (AvPAL*). AvPAL* is used as a therapeutic to treat Phenylketonuria (PKU), a rare autosomal recessive metabolic disorder. Among the three species tested, LGG showed the highest PAL activity followed by L. lactis. Next, we attempted to overcome mass transfer limitation in whole-cell biocatalysts in two ways - expression of heterologous transporters and treatment with different chemical surfactants. Engineered strains expressing heterologous transporters exhibited approximately 3-4-fold increased PAL activity, while chemical treatment did not improve reaction rates. This work highlights the challenges and advances in realizing the potential of LAB as biotherapeutics. Impact StatementOral delivery of phenylalanine ammonia-lyase (PAL) using engineered probiotics is a promising therapeutic strategy to treat Phenylketonuria (PKU). Although PAL expression has been reported in probiotic strains of Limosilactobacillus reuteri, Lactococcus lactis, and E. coli, a systematic comparison of lactic acid bacteria (LAB) is underexplored. This study explores the potential of multiple LAB as hosts for PAL expression and investigates strategies to improve whole cell enzymatic activity. The findings from this study provide a foundation for implementing LAB-based delivery of PAL and indicate an important step towards development of probiotic platform for PKU management.

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Choudhury, D., Mays, Z. J., Nair, N. U.. 2026-03-20. Substrate transport limits phenylalanine ammonia-lyase activity in engineered Lacticaseibacillus rhamnosus GG. https://doi.org/10.64898/2026.03.19.713057

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