Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, we engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pah PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, we found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Our results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.

GCN4 encodes a transcriptional activator of amino acid-biosynthetic genes in Saccharomyces cerevisiae that is regulated at the translational level by upstream open reading frames (uORFs) in its mRNA leader. uORF4 (counting from the 5' end) is sufficient to repress GCN4 under nonstarvation conditions; uORF1 is required to overcome the inhibitory effect of uORF4 and stimulate GCN4 translation in amino acid-starved cells. Insertions of sequences with the potential to form secondary structure around uORF4 abolish derepression, indicating that ribosomes reach GCN4 by traversing uORF4 sequences rather than by binding internally to the GCN4 start site. By showing that wild-type regulation occurred even when uORF4 was elongated to overlap GCN4 by 130 nucleotides, we provide strong evidence that those ribosomes which translate GCN4 do so by ignoring the uORF4 AUG start codon. This conclusion is in accord with the fact that translation of a uORF4-lacZ fusion was lower in a derepressed gcdl mutant than in a nonderepressible gcn2 strain. We also show that increasing the distance between uORF1 and uORF4 to the wild-type spacing that separates uORFl from GCN4 specifically impaired the ability of uORF1 to derepress GCN4 translation. As expected, this alteration led to increased uORF4-lacZ translation in gcdl cells. Our results suggest that under starvation conditions, a substantial fraction of ribosomes that translate uORFl fail to reassemble the factors needed for reinitiation by the time they scan to uORF4, but become competent to reinitiate after scanning the additional sequences to GCN4. Under nonstarvation conditions, ribosomes would recover more rapidly from uORFl translation, causing them all to reinitiate at uORF4 rather than at GCN4.The GCN4 protein of the yeast Saccharomyces cerevisiae is a trahscriptional activator of more than 30 genes involved in the biosynthesis of 10 different amino acids. In response to amino acid starvation, transcription of these genes is stimulated because the rate of GCN4 protein synthesis increases under these conditions. GCN4 expression is regulated by amino acid availability through a translational control mechanism involving four short upstream open reading frames (uORFs) in the leader of GCN4 mRNA. A subset of these uORFs strongly inhibit translation initiation at GCN4 under nonstarvation conditions, and this inhibitory effect is overcome when cells are starved for an amino acid (reviewed in reference 11). Translational repression of GCN4 by the uORFs is dependent on negative regulators encoded by GCD genes. In addition to regulating GCN4 expression, it appears that GCD gene products carry out essential cellular functions, and evidence is accumulating that these functions are involved with the initiation of general protein synthesis (11,35,37). Positive regulators encoded by GCN2 and GCN3 are required for increased translation of GCN4 mRNA under starvation conditions, and these factors are thought to function by antagonizing one or more of the negative-acting GCD proteins (11).Numerous...

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The intestine is a major source of systemic ammonia (NH3); thus, capturing part of gut NH3 may mitigate disease symptoms in conditions of hyperammonemia such as urea cycle disorders and hepatic encephalopathy. As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic Escherichia coli Nissle 1917 to create strain SYNB1020 that converts NH3 to l-arginine (l-arg). We up-regulated arginine biosynthesis in SYNB1020 by deleting a negative regulator of l-arg biosynthesis and inserting a feedback-resistant l-arg biosynthetic enzyme. SYNB1020 produced l-arg and consumed NH3 in an in vitro system. SYNB1020 reduced systemic hyperammonemia, improved survival in ornithine transcarbamylase–deficient spfash mice, and decreased hyperammonemia in the thioacetamide-induced liver injury mouse model. A phase 1 clinical study was conducted including 52 male and female healthy adult volunteers. SYNB1020 was well tolerated at daily doses of up to 1.5 × 1012 colony-forming units administered for up to 14 days. A statistically significant dose-dependent increase in urinary nitrate, plasma 15N-nitrate (highest dose versus placebo, P = 0.0015), and urinary 15N-nitrate was demonstrated, indicating in vivo SYNB1020 activity. SYNB1020 concentrations reached steady state by the second day of dosing, and excreted cells were alive and metabolically active as evidenced by fecal arginine production in response to added ammonium chloride. SYNB1020 was no longer detectable in feces 2 weeks after the last dose. These results support further clinical development of SYNB1020 for hyperammonemia disorders including urea cycle disorders and hepatic encephalopathy.

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