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A platform for distributed production of synthetic nitrated proteins in live bacteria

Nature Chemical Biology (2023)Cite this article

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Abstract

The incorporation of the nonstandard amino acid para-nitro-l-phenylalanine (pN-Phe) within proteins has been used for diverse applications, including the termination of immune self-tolerance. However, the requirement for the provision of chemically synthesized pN-Phe to cells limits the contexts where this technology can be harnessed. Here we report the construction of a live bacterial producer of synthetic nitrated proteins by coupling metabolic engineering and genetic code expansion. We achieved the biosynthesis of pN-Phe in Escherichia coli by creating a pathway that features a previously uncharacterized nonheme diiron N-monooxygenase, which resulted in pN-Phe titers of 820 ± 130 µM after optimization. After we identified an orthogonal translation system that exhibited selectivity toward pN-Phe rather than a precursor metabolite, we constructed a single strain that incorporated biosynthesized pN-Phe within a specific site of a reporter protein. Overall, our study has created a foundational technology platform for distributed and autonomous production of nitrated proteins.

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Fig. 1: De novo biosynthesis and site-specific incorporation of pN-Phe.
Fig. 2: Initial characterization for de novo synthesis pathway.
Fig. 3: Metabolic engineering for the synthesis of pN-Phe in E. coli.
Fig. 4: Bioprospecting of nonheme diiron monooxygenases for activity on pA-Pyr and pA-Phe.
Fig. 5: Aminoacyl-tRNA synthetase screening for incorporation of pN-Phe.
Fig. 6: Integration of the pN-Phe biosynthesis pathway with orthogonal MjTyrRS/tRNA for de novo biosynthesis and incorporation.

Data availability

The datasets generated during and/or analyzed during the current study are contained in the published article (and its Supplementary Information) and are publicly accessible via cited repositories or are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Wu, N., Deiters, A., Cropp, T. A., King, D. & Schultz, P. G. A genetically encoded photocaged amino acid. J. Am. Chem. Soc. 126, 14306–14307 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Neumann, H., Hazen, J. L., Weinstein, J., Mehl, R. A. & Chin, J. W. Genetically encoding protein oxidative damage. J. Am. Chem. Soc. 130, 4028–4033 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Tsao, M. L., Summerer, D., Ryu, Y. & Schultz, P. G. The genetic incorporation of a distance probe into proteins in Escherichia coli. J. Am. Chem. Soc. 128, 4572–4573 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Jackson, J. C., Duffy, S. P., Hess, K. R. & Mehl, R. A. Improving nature’s enzyme active site with genetically encoded unnatural amino acids. J. Am. Chem. Soc. 128, 11124–11127 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Grünewald, J. et al. Immunochemical termination of self-tolerance. Proc. Natl Acad. Sci. USA 105, 11276–11280 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Grünewald, J. et al. Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids. Proc. Natl Acad. Sci. USA 106, 4337–4342 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Gauba, V. et al. Loss of CD4 T-cell-dependent tolerance to proteins with modified amino acids. Proc. Natl Acad. Sci. USA 108, 12821–12826 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Shorter, J. PATAI’S Chemistry of Functional Groups (John Wiley & Sons, 2009). https://doi.org/10.1002/9780470682531.pat0081

  9. Tian, H. et al. Nitrated T helper cell epitopes enhance the immunogenicity of HER2 vaccine and induce anti-tumor immunity. Cancer Lett. 430, 79–87 (2018).

    Article  CAS  PubMed  Google Scholar 

  10. Tian, H. et al. PDL1-targeted vaccine exhibits potent antitumor activity by simultaneously blocking PD1/PDL1 pathway and activating PDL1-specific immune responses. Cancer Lett. 476, 170–182 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Li, F. et al. A new vaccine targeting RANKL, prepared by incorporation of an unnatural amino acid into RANKL, prevents OVX-induced bone loss in mice. Biochem. Biophys. Res. Commun. 499, 648–654 (2018).

    Article  CAS  PubMed  Google Scholar 

  12. Völler, J. S. & Budisa, N. Coupling genetic code expansion and metabolic engineering for synthetic cells. Curr. Opin. Biotechnol. 48, 1–7 (2017).

    Article  PubMed  Google Scholar 

  13. Dickey, R. M., Forti, A. M. & Kunjapur, A. M. Advances in engineering microbial biosynthesis of aromatic compounds and related compounds. Bioresour. Bioprocess. 8, 91 (2021).

    Article  Google Scholar 

  14. Ding, C., Ma, J., Dong, Q. & Liu, Q. Live bacterial vaccine vector and delivery strategies of heterologous antigen: a review. Immunol. Lett. 197, 70–77 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. van Bloois, E., Winter, R. T., Kolmar, H. & Fraaije, M. W. Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol. 29, 79–86 (2011).

    Article  PubMed  Google Scholar 

  16. Wang, S. et al. Salmonella vaccine vectors displaying delayed antigen synthesis in vivo to enhance immunogenicity. Infect. Immun. 78, 3969–3980 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mehl, R. A. et al. Generation of a bacterium with a 21 amino acid genetic code. J. Am. Chem. Soc. 125, 935–939 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Chen, Y. et al. Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation. Nat. Commun. 13, 5434 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kim, S., Sung, B. H., Kim, S. C. & Lee, H. S. Genetic incorporation of l -dihydroxyphenylalanine (DOPA) biosynthesized by a tyrosine phenol-lyase. Chem. Commun. 54, 3002–3005 (2018).

    Article  CAS  Google Scholar 

  20. Zuo, R. & Ding, Y. Direct aromatic nitration system for synthesis of nitrotryptophans in Escherichia coli. ACS Synth. Biol. 8, 857–865 (2019).

    Article  CAS  PubMed  Google Scholar 

  21. Chen, Y. et al. Creation of bacterial cells with 5-hydroxytryptophan as a 21st amino acid building block. Chem 6, 2717–2727 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Marchand, J. A. et al. Discovery of a pathway for terminal-alkyne amino acid biosynthesis. Nature 567, 420–424 (2019).

    Article  CAS  PubMed  Google Scholar 

  23. Waldman, A. J., Ng, T. L., Wang, P. & Balskus, E. P. Heteroatom–heteroatom bond formation in natural product biosynthesis. Chem. Rev. 117, 5784–5863 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Masuo, S., Zhou, S., Kaneko, T. & Takaya, N. Bacterial fermentation platform for producing artificial aromatic amines. Sci. Rep. 6, 25764 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chen, Y. et al. A noncanonical amino acid-based relay system for site-specific protein labeling. Chem. Commun. 54, 7187–7190 (2018).

    Article  CAS  Google Scholar 

  26. Schaffer, J. E., Reck, M. R., Prasad, N. K. & Wencewicz, T. A. β-Lactone formation during product release from a nonribosomal peptide synthetase. Nat. Chem. Biol. 13, 737–744 (2017).

    Article  CAS  PubMed  Google Scholar 

  27. Scott, T. A., Heine, D., Qin, Z. & Wilkinson, B. An l-threonine transaldolase is required for l-threo-β-hydroxy-α-amino acid assembly during obafluorin biosynthesis. Nat. Commun. 8, 15935 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chanco, E., Choi, Y. S., Sun, N., Vu, M. & Zhao, H. Characterization of the N-oxygenase AurF from Streptomyces thioletus. Bioorg. Med. Chem. 22, 5569–5577 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Lu, H., Chanco, E. & Zhao, H. CmlI is an N-oxygenase in the biosynthesis of chloramphenicol. Tetrahedron 68, 7651–7654 (2012).

    Article  CAS  Google Scholar 

  30. Finnigan, W., Hepworth, L. J., Flitsch, S. L. & Turner, N. J. RetroBioCat as a computer-aided synthesis planning tool for biocatalytic reactions and cascades. Nat. Catal. 4, 98–104 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hadadi, N., Hafner, J., Shajkofci, A., Zisaki, A. & Hatzimanikatis, V. ATLAS of biochemistry: a repository of all possible biochemical reactions for synthetic biology and metabolic engineering studies. ACS Synth. Biol. 5, 1155–1166 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Rau, J. & Stolz, A. Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent azo reductases. Appl. Environ. Microbiol. 69, 3448–3455 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Onuffer, J. J., Ton, B. T., Klement, I. & Kirsch, J. F. The use of natural and unnatural amino acid substrates to define the substrate specificity differences of Escherichia coli aspartate and tyrosine aminotransferases. Protein Sci. 4, 1743–1749 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kunjapur, A. M., Tarasova, Y. & Prather, K. L. J. Synthesis and accumulation of aromatic aldehydes in an engineered strain of Escherichia coli. J. Am. Chem. Soc. 136, 11644–11654 (2014).

    Article  CAS  PubMed  Google Scholar 

  35. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Seong Choi, Y., Zhang, H., Brunzelle, J. S., Nair, S. K. & Zhao, H. In vitro reconstitution and crystal structure of p-aminobenzoate N-oxygenase (AurF) involved in aureothin biosynthesis. Proc. Natl Acad. Sci. USA 105, 6858–6863 (2008).

    Article  Google Scholar 

  38. Young, D. D. et al. An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity. Biochemistry 50, 1894–1900 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Kunjapur, A. M. et al. Engineering posttranslational proofreading to discriminate nonstandard amino acids. Proc. Natl Acad. Sci. USA 115, 619–624 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Blizzard, R. J. et al. Ideal bioorthogonal reactions using a site-specifically encoded tetrazine amino acid. J. Am. Chem. Soc. 137, 10044–10047 (2015).

    Article  CAS  PubMed  Google Scholar 

  41. Wang, L., Brock, A. & Schultz, P. G. Adding l-3-(2-naphthyl)alanine to the genetic code of E. coli. J. Am. Chem. Soc. 124, 1836–1837 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Wang, L., Zhang, Z., Brock, A. & Schultz, P. G. Addition of the keto functional group to the genetic code of Escherichia coli. Proc. Natl Acad. Sci. USA 100, 56–61 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Wörsdörfer, B. et al. Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer. J. Am. Chem. Soc. 135, 8585–8593 (2013).

    Article  PubMed  Google Scholar 

  44. Cui, L. & Shearwin, K. E. Methods in Molecular Biology Vol. 1472, pp. 139–155 (Humana Press, 2017).

  45. Wannier, T. M. et al. Improved bacterial recombineering by parallelized protein discovery. Proc. Natl Acad. Sci. USA 117, 13689–13698 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kunjapur, A. M., Hyun, J. C. & Prather, K. L. J. Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway. Micro. Cell Fact. 15, 61 (2016).

    Article  Google Scholar 

  47. Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).

    Article  CAS  PubMed  Google Scholar 

  48. Guo, Y. Y. et al. Molecular mechanism of azoxy bond formation for azoxymycins biosynthesis. Nat. Commun. 10, 4420 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Gerlt, J. A. et al. Enzyme function initiative-enzyme similarity tool (EFI-EST): a web tool for generating protein sequence similarity networks. Biochim. Biophys. Acta 1854, 1019–1037 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Kretsch, A. et al. Discovery of (dihydro)pyrazine N-oxides via genome mining in Pseudomonas. Org. Lett. 20, 4791–4795 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Platter, E., Lawson, M., Marsh, C. & Sazinsky, M. H. Characterization of a non-ribosomal peptide synthetase-associated diiron arylamine N-oxygenase from Pseudomonas syringae pv. phaseolicola. Arch. Biochem. Biophys. 508, 39–45 (2011).

    Article  CAS  PubMed  Google Scholar 

  52. Indest, K., Eberly, J. & Hancock, D. Expression and characterization of an N-oxygenase from Rhodococcus jostii RHAI. J. Gen. Appl. Microbiol. 61, 217–223 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Cortina, N. S., Revermann, O., Krug, D. & Müller, R. Identification and characterization of the althiomycin biosynthetic gene cluster in Myxococcus xanthus DK897. ChemBioChem 12, 1411–1416 (2011).

    Article  CAS  PubMed  Google Scholar 

  54. Jenul, C. et al. Biosynthesis of fragin is controlled by a novel quorum sensing signal. Nat. Commun. 9, 1297 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Tsutsumi, H. et al. Unprecedented cyclization catalyzed by a cytochrome P450 in benzastatin biosynthesis. J. Am. Chem. Soc. 140, 6631–6639 (2018).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank the Mass Spectrometry Facility at the University of Delaware for the mass spectrometry analysis that is supported by the National Institute of General Medical Sciences or the National Institutes of Health under award P20GM104316 and Y. Yu and P.N. Asare-Okai in particular for their assistance. We acknowledge support from the following funding sources: The National Science Foundation (NSF CBET-2032243, to A.M.K.), University of Delaware Start-Up Funds (to A.M.K.), the Mort Collins Foundation (to N.D.B.) and minor research support as part of the Center for Plastics Innovation, an Energy Frontier Research Center funded by the U.S. Department of Energy (DOE), Office of Science, Basic Energy Sciences, under award DE-SC0021166 (to A.M.K.). We are also grateful to the American Institute of Chemical Engineers for their support of this concept through the 2021 Langer Prize for Innovation and Entrepreneurial Excellence (to A.M.K.). We also thank M. Jones for valuable experimental troubleshooting suggestions for this work.

Author information

Authors and Affiliations

  1. Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, DE, USA

    Neil D. Butler, Sabyasachi Sen, Minwei Lin & Aditya M. Kunjapur

  2. Systems, Synthetic, and Physical Biology Program, Rice University, Houston, TX, USA

    Lucas B. Brown

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Contributions

A.M.K. conceived and supervised the study; N.D.B. designed and performed all experiments, analyzed data, prepared figures and wrote the manuscript; M.L. aided with molecular cloning; S.S. cloned the N-oxygenase library and confirmed expression and L.B.B. cloned the MjTyrRS variants tested in this study.

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Correspondence to Aditya M. Kunjapur.

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Competing interests

N.D.B. and A.M.K. are co-inventors on a filed patent application related to this work that has now been transferred to a commercial entity cofounded by the authors (Nitro Biosciences). A.M.K. also serves on the Scientific Advisory Board of Wild Microbes. The remaining authors declare no competing interests.

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Nature Chemical Biology thanks Jorge Marchand and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Tables 1–10 and Supplementary Figs. 1–19.

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Supplementary Data

Supporting data for Supplementary Figs. 2, 10–13, 15, 16 and 18.

Source data

Source Data Fig. 2

Peak areas obtained from HPLC.

Source Data Fig. 3

Peak areas obtained from HPLC.

Source Data Fig. 4

Peak areas obtained from HPLC.

Source Data Fig. 5

Raw plate reader data.

Source Data Fig. 6

Raw plate reader data and peak areas obtained from HPLC.

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Butler, N.D., Sen, S., Brown, L.B. et al. A platform for distributed production of synthetic nitrated proteins in live bacteria. Nat Chem Biol (2023). https://doi.org/10.1038/s41589-023-01338-x

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