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

doi:10.1038/s41589-023-01338-x

. 2023 Jul;19(7):911-920.

doi: 10.1038/s41589-023-01338-x. Epub 2023 May 15.

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

Neil D Butler¹, Sabyasachi Sen¹, Lucas B Brown², Minwei Lin¹, Aditya M Kunjapur³

Affiliations

¹ Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, DE, USA.
² Systems, Synthetic, and Physical Biology Program, Rice University, Houston, TX, USA.
³ Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, DE, USA. kunjapur@udel.edu.

PMID: 37188959
DOI: 10.1038/s41589-023-01338-x

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

Neil D Butler et al. Nat Chem Biol. 2023 Jul.

. 2023 Jul;19(7):911-920.

doi: 10.1038/s41589-023-01338-x. Epub 2023 May 15.

Authors

Neil D Butler¹, Sabyasachi Sen¹, Lucas B Brown², Minwei Lin¹, Aditya M Kunjapur³

Affiliations

¹ Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, DE, USA.
² Systems, Synthetic, and Physical Biology Program, Rice University, Houston, TX, USA.
³ Department of Chemical & Biomolecular Engineering, University of Delaware, Newark, DE, USA. kunjapur@udel.edu.

PMID: 37188959
DOI: 10.1038/s41589-023-01338-x

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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Cited by

Cellular Site-Specific Incorporation of Noncanonical Amino Acids in Synthetic Biology.
Niu W, Guo J. Niu W, et al. Chem Rev. 2024 Sep 25;124(18):10577-10617. doi: 10.1021/acs.chemrev.3c00938. Epub 2024 Aug 29. Chem Rev. 2024. PMID: 39207844 Review.
Nitro Biosciences:: Enhancing immune response via an expanded genetic code.
Butler N, Kunjapur A. Butler N, et al. Dela J Public Health. 2023 Nov 30;9(4):24-25. doi: 10.32481/djph.2023.11.007. eCollection 2023 Nov. Dela J Public Health. 2023. PMID: 38173964 Free PMC article.

References

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[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). - PubMed

[2] 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). - PubMed

[3] 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). - PubMed

[4] 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). - PubMed

[5] 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). - PubMed

[6] 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). - PubMed

[7] 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). - PubMed

[8] 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). - PubMed

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

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

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

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

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