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.

将非标准氨基酸对硝基-L-苯丙氨酸 (pN-Phe) 掺入蛋白质中已被用于多种应用，包括终止免疫自我耐受。然而，向细胞提供化学合成的 pN-Phe 的要求限制了该技术的应用范围。在这里，我们报告了通过耦合代谢工程和遗传密码扩展来构建合成硝化蛋白质的活细菌生产者。我们通过创建一条以前未表征的非血红素二铁N -单加氧酶为特征的途径，在大肠杆菌中实现了 pN-Phe 的生物合成，优化后 pN-Phe 滴度为 820 ± 130 µM。在我们确定了一个对 pN-Phe 而不是前体代谢物表现出选择性的正交翻译系统后，我们构建了一个单一菌株，将生物合成的 pN-Phe 整合到报告蛋白的特定位点内。总的来说，我们的研究为硝化蛋白质的分布式和自主生产创建了一个基础技术平台。