Article | Published:

Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria

Nature Biotechnology volume 36, pages 857864 (2018) | Download Citation

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

BioProject

Referenced accessions

GenBank/EMBL/DDBJ

NCBI Reference Sequence

References

  1. 1.

    , , , & Pathogenesis of cognitive dysfunction in phenylketonuria: review of hypotheses. Mol. Genet. Metab. 99 (Suppl. 1), S86–S89 (2010).

  2. 2.

    et al. Are neuropsychological impairments in children with early-treated phenylketonuria (PKU) related to white matter abnormalities or elevated phenylalanine levels? Dev. Neuropsychol. 32, 645–668 (2007).

  3. 3.

    et al. Psychiatric symptoms in adults with phenylketonuria. Mol. Genet. Metab. 108, 155–160 (2013).

  4. 4.

    et al. The impact of phenylalanine levels on cognitive outcomes in adults with phenylketonuria: Effects across tasks and developmental stages. Neuropsychology 31, 242–254 (2017).

  5. 5.

    et al. Phenylalanine hydroxylase deficiency: diagnosis and management guideline. Genet. Med. 16, 188–200 (2014).

  6. 6.

    et al. Work activity and phenylalanine levels in a population of young adults with classic PKU. Med. Lav. 108, 118–122 (2017).

  7. 7.

    et al. Long-term safety and efficacy of sapropterin: the PKUDOS registry experience. Mol. Genet. Metab. 114, 557–563 (2015).

  8. 8.

    et al. Long-term developmental progression in infants and young children taking sapropterin for phenylketonuria: a two-year analysis of safety and efficacy. Genet. Med. 17, 365–373 (2015).

  9. 9.

    & Sapropterin dihydrochloride for phenylketonuria. Cochrane Database Syst. Rev. 16, CD008005 (2010).

  10. 10.

    et al. Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria. PLoS One 12, e0173269 (2017).

  11. 11.

    et al. Preclinical evaluation of multiple species of PEGylated recombinant phenylalanine ammonia lyase for the treatment of phenylketonuria. Proc. Natl. Acad. Sci. USA 105, 20894–20899 (2008).

  12. 12.

    & Effects of oral administration of artificial cells immobilized phenylalanine ammonia-lyase on intestinal amino acids of phenylketonuric rats. Biomater. Artif. Cells Artif. Organs 17, 161–181 (1989).

  13. 13.

    , & A new theory of enterorecirculation of amino acids and its use for depleting unwanted amino acids using oral enzyme-artificial cells, as in removing phenylalanine in phenylketonuria. Artif. Cells Blood Substit. Immobil. Biotechnol. 23, 1–21 (1995).

  14. 14.

    Therapeutic applications of polymeric artificial cells. Nat. Rev. Drug Discov. 4, 221–235 (2005).

  15. 15.

    , & Synthetic biology moving into the clinic. Science 333, 1248–1252 (2011).

  16. 16.

    et al. A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase. Proc. Natl. Acad. Sci. USA 96, 2339–2344 (1999).

  17. 17.

    , & Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU. PLoS One 12, e0176286 (2017).

  18. 18.

    Escherichia coli strain Nissle 1917-from bench to bedside and back: history of a special Escherichia coli strain with probiotic properties. FEMS Microbiol. Lett. 363, fnw212 (2016).

  19. 19.

    Clinical use of E. coli Nissle 1917 in inflammatory bowel disease. Inflamm. Bowel Dis. 14, 1012–1018 (2008).

  20. 20.

    , , , & Survival of the probiotic Escherichia coli Nissle 1917 (EcN) in the gastrointestinal tract given in combination with oral mesalamine to healthy volunteers. Inflamm. Bowel Dis. 16, 256–262 (2010).

  21. 21.

    et al. Translational development of microbiome-based therapeutics: kinetics of E. coli Nissle and engineered strains in humans and nonhuman primates. Clin. Transl. Sci. 11, 200–207 (2018).

  22. 22.

    et al. Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models. Nat. Commun. 8, 15028 (2017).

  23. 23.

    et al. Programmable probiotics for detection of cancer in urine. Sci. Transl. Med. 7, 289ra84 (2015).

  24. 24.

    , & Genetically engineered Escherichia coli Nissle 1917 synbiotics reduce metabolic effects induced by chronic consumption of dietary fructose. PLoS One 11, e0164860 (2016).

  25. 25.

    et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J. Clin. Invest. 124, 3391–3406 (2014).

  26. 26.

    et al. Engineered probiotic for the inhibition of salmonella via tetrathionate-induced production of Microcin H47. ACS Infect. Dis. 4, 39–45 (2018).

  27. 27.

    , & The gene stlA encodes a phenylalanine ammonia-lyase that is involved in the production of a stilbene antibiotic in Photorhabdus luminescens TT01. Microbiology 151, 2543–2550 (2005).

  28. 28.

    & A modern view of phenylalanine ammonia lyase. Biochem. Cell Biol. 85, 273–282 (2007).

  29. 29.

    et al. A study of AroP-PheP chimeric proteins and identification of a residue involved in tryptophan transport. J. Bacteriol. 182, 2207–2217 (2000).

  30. 30.

    , , & Structure-function relationships in L-amino acid deaminase, a flavoprotein belonging to a novel class of biotechnologically relevant enzymes. J. Biol. Chem. 291, 10457–10475 (2016).

  31. 31.

    et al. Two-Step production of phenylpyruvic acid from L-phenylalanine by growing and resting cells of engineered Escherichia coli: process optimization and kinetics modeling. PLoS One 11, e0166457 (2016).

  32. 32.

    et al. Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches. Appl. Microbiol. Biotechnol. 99, 8391–8402 (2015).

  33. 33.

    et al. Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract of living mice with spatial and spectral EPR imaging. Proc. Natl. Acad. Sci. USA 96, 4586–4591 (1999).

  34. 34.

    , & Purification and characterization of an L-amino acid deaminase used to prepare unnatural amino acids. J. Mol. Catal. 11, 795–803 (2001).

  35. 35.

    , , , , & Heterologous expression and characterization of L-amino acid deaminase from Proteus mirabilis in Escherichia coli. Chin. J. Biotechnol. 24, 21–29 (2008).

  36. 36.

    , , , & Translational regulation of gene expression by an anaerobically induced small non-coding RNA in Escherichia coli. J. Biol. Chem. 285, 10690–10702 (2010).

  37. 37.

    & Reprogramming of anaerobic metabolism by the FnrS small RNA. Mol. Microbiol. 75, 1215–1231 (2010).

  38. 38.

    et al. Control of FNR function of Escherichia coli by O2 and reducing conditions. J. Mol. Microbiol. Biotechnol. 4, 263–268 (2002).

  39. 39.

    et al. Respiration of Escherichia coli in the mouse intestine. Infect. Immun. 75, 4891–4899 (2007).

  40. 40.

    , , & Different modes of diaminopimelate synthesis and their role in cell wall integrity: a study with Corynebacterium glutamicum. J. Bacteriol. 180, 3159–3165 (1998).

  41. 41.

    , , & GeneGuard: A modular plasmid system designed for biosafety. ACS Synth. Biol. 4, 307–316 (2015).

  42. 42.

    et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55–60 (2015).

  43. 43.

    , & Building-in biosafety for synthetic biology. Microbiology 159, 1221–1235 (2013).

  44. 44.

    , , & Mouse models of human phenylketonuria. Genetics 134, 1205–1210 (1993).

  45. 45.

    , & Phenylketonuria: an inborn error of phenylalanine metabolism. Clin. Biochem. Rev. 29, 31–41 (2008).

  46. 46.

    , & Post-mortem survival of hippuric acid formation in rat and human cadaver tissue samples. Xenobiotica 6, 275–280 (1976).

  47. 47.

    & Phenylalanine ammonia lyase in the management of phenylketonuria: the relationship between ingested cinnamate and urinary hippurate in humans. Res. Commun. Chem. Pathol. Pharmacol. 35, 275–282 (1982).

  48. 48.

    Dietary Guidelines should reflect new understandings about adult protein needs. Nutr. Metab. (Lond.) 6, 12 (2009).

  49. 49.

    et al. Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol. 421, 171–199 (2007).

  50. 50.

    , , , & Recombineering: genetic engineering in bacteria using homologous recombination. Curr. Protoc. Mol. Biol. 106, 1.16.11–1.16.39 (2014).

  51. 51.

    Experiments in Molecular Genetics (Cold Spring Harbor Laboratory Press, 1972).

Download references

Acknowledgements

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.

Author information

Author notes

    • Sarah E Rowe

    Present address: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Affiliations

  1. Synlogic Inc., Cambridge, Massachusetts, USA.

    • Vincent M Isabella
    • , Binh N Ha
    • , Mary Joan Castillo
    • , David J Lubkowicz
    • , Sarah E Rowe
    • , Yves A Millet
    • , Cami L Anderson
    • , Ning Li
    • , Adam B Fisher
    • , Kip A West
    • , Philippa J Reeder
    • , Munira M Momin
    • , Christopher G Bergeron
    • , Sarah E Guilmain
    • , Paul F Miller
    • , Caroline B Kurtz
    •  & Dean Falb

Authors

  1. Search for Vincent M Isabella in:

  2. Search for Binh N Ha in:

  3. Search for Mary Joan Castillo in:

  4. Search for David J Lubkowicz in:

  5. Search for Sarah E Rowe in:

  6. Search for Yves A Millet in:

  7. Search for Cami L Anderson in:

  8. Search for Ning Li in:

  9. Search for Adam B Fisher in:

  10. Search for Kip A West in:

  11. Search for Philippa J Reeder in:

  12. Search for Munira M Momin in:

  13. Search for Christopher G Bergeron in:

  14. Search for Sarah E Guilmain in:

  15. Search for Paul F Miller in:

  16. Search for Caroline B Kurtz in:

  17. Search for Dean Falb in:

Contributions

V.M.I., D.J.L., S.E.R., N.L. and A.B.F. were responsible for strain construction and performance of in vitro experiments. P.J.R. conducted the analysis of in vitro promoter activity. V.M.I. and C.L.A. analyzed the data. B.N.H. performed mouse experiments. M.J.C. and Y.A.M. performed mass spectrometry analysis. M.M.M. and C.G.B. grew cells in a bioreactor. V.M.I., K.A.W., P.F.M., C.K., S.E.G. and D.F. helped supervise the project. All of the authors helped to prepare the manuscript.

Competing interests

Authors hold stock in Synlogic and may gain or lose financially through publication. For information or material, please direct correspondence to vincent@synlogictx.com.

Corresponding author

Correspondence to Vincent M Isabella.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Tables

    Supplementary Tables 1–5

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt.4222

Further reading