Biology subjects

Mays, Z. J.

Publications and source records attributed to Mays, Z. J..

3 recordsLinked to original sources

bioRxiv2026

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

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.

synthetic biology↗

bioRxiv2020

A quantitative model for oral administration of living therapeutics

As medicine shifts toward precision-based and personalized therapeutics, utilizing more complex biomolecules to treat increasingly difficult and rare conditions, microorganisms provide an avenue for realizing the production and processing necessary for novel drug pipelines. More so, probiotic microbes can be co-opted to deliver therapeutics by oral administration as living drugs, able to survive and safely transit the digestive tract. As living therapeutics are in their nascency, traditional pharmacokinetic-pharmacodynamic (PK-PD) models for evaluating drug candidates are not appropriate for this novel platform. Using a living therapeutic in late-stage clinical development for phenylketonuria (PKU) as a case study, we adapt traditional oral drug delivery models to properly evaluate and inform the engineering of living therapeutics. We develop the adapted for living therapeutics compartmental absorption and transit (ALT-CAT) model to provide metrics for drug efficacy across nine age groups of PKU patients and evaluate model parameters that are influenced by patient physiology, microbe selection and therapeutic production, and dosing formulations. ImportanceThis work describes a kinetic model to study the behavior of orally delivered living therapeutics. Such therapeutics are becoming increasingly relevant and are an exciting mode of drug delivery that stems from the growing interest through the convergence of advances in synthetic biology of probiotics and gut microbes as well as microbiome science. In particular, this work describes the development of a mathematical framework (pharmacokinetic-pharmacodynamic, PK-PD) called ALT-CAT to model the behavior of orally delivered engineered bacteria that act as living therapeutics by adapting similar methods that have been developed and widely-used for small molecular drug delivery and absorption.

bioengineering↗

bioRxiv2020

Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities

There is broad interest in engineering phenylalanine ammonia-lyase (PAL) for its biocatalytic applications in industry (fine-chemicals and natural product synthesis) and medicine (phenylketoruria/PKU and cancer treatment). While site-specific mutagenesis has been employed to improve PAL stability or substrate specificity, a more comprehensive mutational landscape has yet to be explored for this class of enzymes. Here, we report development of a directed evolution technique to engineer PAL enzymes. Central to this approach is a high-throughput enrichment that couples E. coli growth to PAL activity. Using the clinically-relevant PAL from Anabaena variabilis, which is used on the formulation of pegvaliase for PKU therapy, we identified mutations at residues previously unknown as relevant for function that increase turnover frequency almost twofold after only a single round of engineering. This work demonstrates the power our technique for ammonia-lyase enzyme engineering.

bioengineering↗