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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., PKU patients have elevated phenylalanine levels which can result in neurological impairment. Here the authors utilize biosensor-based ultra-high-throughput screening to optimize PAL activity in a synthetic biotic platform for improved in vivo performance.

Transcription, the first step to gene expression, is a central coordination process in all living matter. Besides a plethora of regulatory mechanisms, the promoter architecture sets the foundation of expression strength, timing and the potential for further regulatory modulation. In this study, we investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. Combining transcriptomics data analysis and standardized synthetic promoter libraries, we exclude effects of specific promoter sequence contexts. Analysis of promoter activity shows a strong variance with spacer length and spacer sequence composition. A detailed study of the spacer sequence composition under selective conditions reveals a, RNAP binding enhancing but expression damping, extension to the –10 region. Using physiological changes in DNA supercoiling levels, we link promoter supercoiling sensitivity to overall spacer GC-content. Time-resolved promoter activity screens, only possible with a novel mild treatment approach, reveal strong promoter timing potentials solely based on DNA supercoiling sensitivity in the absence of regulatory sites or alternative sigma factors.