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

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.