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 Pahenu2/enu2 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.
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We thank J. Collins, M. Charbonneau, A. Brennan and E. Wolffe for their discussions and comments on the manuscript, and B. Peters for assistance with graphics.
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Integrated supplementary information
Enzymatic reactions with Phe and associated metabolites. (a) PAL or LAAD activity results in the formation of trans-cinnamate (TCA) or phenylpyruvate (PP) respectively. (b) The SYNB1618-specific metabolite trans-cinnamate is converted systemically in the host to hippurate, which is excreted and may be measured in urine.
Supplementary Figure 2 Promoter activity of a Pfnrs-lacZ fusion in aerobic and anaerobic conditions in E. coli Nissle.
An E. coli Nissle strain containing a low copy plasmid with a PfnrS-lacZ transcriptional promoter fusionwas grown overnight with vigorous aerobic shaking and back-diluted 1:100 into fresh culture media in a flask with vigorous shaking. At time 0, the culture was split; half remained shaking and the other half was incubated statically in an anaerobic chamber. At the indicated time points, 1mL of culture was removed and promoter activity was determined by assessing β-galactosidase levels. Anaerobic induction was observed. Data is shown for 2 independent replicate cultures.
An E. coli Nissle strain containing a low copy plasmid with a PfnrS-GFP transcriptional promoter fusion was grown overnight and then used to inoculate 2 mL wells of a BioLector microbioreactor system with the volume of overnight culture indicated (2 replicates each of 10, 100, 150, and 200μL inoculum and 1 replicate of 50μL inoculum). Oxygen transfer rate remained constant over time. Dissolved oxygen content (solid lines) in the culture media was shown to decrease over time with culture growth, with lower inocula resulting in slower consumption of oxygen. Increased GFP detection (right y-axis, dotted lines), and thus PfnrS-mediated expression, was associated with decreasing dissolved oxygen content of the culture media, consistent with PfnrS promoter activation under oxygen-limitation.
Activation of the PfnrS promoter was measured in vivo. (a) Initial in vitro profiling of the Pfnrs promoter was performed by flow cytometry. Aerobic (+O2, left histogram) and anaerobic (-O2, right histogram) cultures were analyzed with a negative control strain of EcN without GFP (red), a positive control strain expressing GFP constitutively (blue), and a strain expressing GFP under control of the PfnrS promoter (green). As expected, the negative control showed no detectable GFP signal in either aerobic or anaerobic conditions, the positive control strain showed equivalent GFP expression in either aerobic or anaerobic conditions, and the PfnrS-GFP fusion strain only expressed GFP under anaerobic conditions (Note: While the constitutive control was expressed from the chromosome, PfnrS-GFP was expressed from a plasmid, which may have led to more variable and higher level expression observed in the historgrams). This experiment was repeated independently with similar results (b) The aerobically grown strains analyzed in (a) were dosed orally to C57BL/6 mice and recovered 4h post-dose by flushing excised cecal tissue with PBS As demonstrated in (a), the PfnrS-GFP fusion strain was off at the time of dosing. Cecal effluents were analyzed by flow cytometry. For each plot, 40,000 counted events are shown. The y-axis represents the SSC and the x-axis the GFP channel. Plot 1 shows analysis of the background and autofluorescence of cecal effluent in a mouse that did not receive cells. Plot 2 shows analysis of cecal effluent from a mouse dosed with control EcN (no GFP), and no fluorescence is observed. Plot 3 shows analysis of cecal effluent after dosing with EcN expressing GFP constitutively, and a GFP fluorescent population is observed and gated on. This gate was applied to all plots. Plot 4 shows analysis of cecal effluent after dosing with EcN containing a PfnrS-GFP fusion that were OFF at the time of dosing. GFP fluorescence was observed in cecal effluent (increased abundance of events counted in the GFP-positive gate). When normalized to CFU counts recovered from the effluent and by subtracting cecal effluent background (Plot 1), the gated populations account for ~76 % (constitutive GFP control) and ~64 % (PfnrS-GFP) of the cells recovered by plating. Though considerable variability exists in CFU counting, especially from biological samples, the conclusion is that a majority of the PfnrS-controlled cells appear to enter the “ON” state post-dosing. This experiment was repeated independently with similar results
To characterize the growth of E. coli Nissle (EcN) and therapeutic candidate SYNB1618, which contains a mutation in the dapA gene, both strains were incubated in LB that did (+) or did not (-) contain diaminopimelic acid (DAP; 100 μg/mL) at 37 °C for 960 minutes under constant shaking. The OD600 was measured every 10 minutes to assess cell growth over time. The average OD ± standard deviation of 6 replicate cultures is plotted for each time point. Data shows that SYNB1618 is unable to grow without the addition of exogenous DAP to the growth media.
Proposed mechanism of enterorecirculation of Phenylalanine. Proteins are continuously replenished in the intestinal lumen though the introduction and digestion of dietary protein and from glandular secretions as well as through turnover of the intestinal epithelial cells. Intestinal proteolysis generates a pool of Phe containing peptides and free Phe which may then be reabsorbed back into the body as they pass down the intestine. Peptides and free Phe may be converted into proteins systemically which may be reintroduced into the intestine through glandular secretions and amino acid reabsorption (see Chang and Lister, 1995).
Supplementary Figure 7 In vivo PAL activity in engineered strains is increased upon co-expression with pheP
ENU2 mice on Phe-deficient diet were housed in metabolic cages and orally gavaged with 3 × 1010 flask-grown cells of E. coli Nissle strains expressing the gene encoding PAL from a plasmid, with or without co-expression of a chromosomally integrated pheP gene (Strains PAL or PAL/PheP respectively, Supplementary Table 3, n = 9) Each dot represents a metabolic cage of 3 mice/cage. Urine was collected for 4h and analyzed for HA content by LC-MS/MS. Bars represent the average urinary HA recovery of the 3 cages.
Supplementary Figure 8 Conversion efficiency of oral trans-cinnamate to urinary hippurate in non-human primates
NHPs (n = 6) were orally administered 13C-trans-cinnamate (13C-TCA) and urine was collected over 6h. Each bar represents a single NHP subject. 13C-Hippurate (13C-HA) was measured in the urine by mass spectroscopy. The percentage of urinary 13C-HA recovered as a function of 13C-TCA administered was calculated and used as a normalization factor for HA recovery in subsequent experiments. This factor accounts for TCA that is not converted to HA or that is lost to incomplete urinary collection, thus allowing a more accurate description of strain activity.
Using LC-MS/MS, serum concentrations of d5-HA (a) and d5-TCA (b) were determined in non-human primates administered d5-Phe and SYNB1618 orally. No detectable d5-HA or d-TCA was detected when d5-Phe was administered in the absence of SYNB1618 (data not shown). The presence of these metabolites demonstrates SYNB1618-specific activity in these animals.
Schematic of clinical candidate SYNB1618. SYNB1618 contains chromosomally inserted genes encoding PheP, a high affinity phenylalanine (Phe) transporter that can bring Phe into the cell, PAL (stlA), which converts Phe into trans-cinnamic acid (TCA), and LAAD (pma), which converts Phe to phenylpyruvate (PP). Regulation of these components is carried out by anaerobic- IPTG-, and L-arabinose-inducible promoters, for activation in the mammalian gut or in vitro. The locations of the genomic modification sites in SYNB1618 are shown, with kbp designation indicating the chromosomal position relative to the 0/5.4 Mb reference marker. The chromosomal origin of replication is shown as a red line. Italicized gene names in parenthesis refer to the upstream and downstream genes surrounding the inserted gene.
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Isabella, V., Ha, B., Castillo, M. et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat Biotechnol 36, 857–864 (2018). https://doi.org/10.1038/nbt.4222
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