Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering
Kristin J. Adolfsen, Isolde Callihan, Catherine Monahan, Per Greisen, James Spoonamore, Munira Momin, Lauren E. Fitch, Mary Joan Castillo, Lindong Weng, Lauren Renaud, Carl J. Weile, Jay H. Konieczka, Teodelinda Mirabella, Andrés Abín-Fuentes, Adam G. Lawrence, Vincent M. Isabella
Abstract
In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic's Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618.