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

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.

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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.

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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.

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Authors and Affiliations

Authors

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.

Corresponding author

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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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 19, 911–920 (2023). https://doi.org/10.1038/s41589-023-01338-x

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