De novo biosynthesis of para-nitro-L-phenylalanine in Escherichia coli

Abstract

Nitroaromatic functional groups can impart valuable properties to chemicals and to biological macromolecules including polypeptides. Para-nitro-L-phenylalanine (pN-Phe) is a nitroaromatic amino acid with uses including immune stimulation and fluorescence quenching. As the chemical synthesis of pN-Phe does not follow green chemistry principles and impedes provision of pN-Phe to engineered bacterial cells in some contexts, we sought to design a de novo biosynthetic pathway for pN-Phe in Escherichia coli. To generate the nitro chemical functional group, we identified natural diiron monooxygenases with measurable in vitro and in vivo activity on envisioned amine-containing precursors of para-amino-L-phenylalanine (pA-Phe) and para-aminophenylpyruvate. By expressing one of these N-oxygenase genes together with previously characterized genes for the biosynthesis of pA-Phe, we achieved the synthesis of pN-Phe from glucose. Through further optimization of the chassis, plasmid constructs, and media conditions, we were able to improve the selectivity of pN-Phe biosynthesis, resulting in a maximum titer of 819 {micro}M in rich defined media under shake-flask conditions. These results provide a foundation for the biosynthesis of related nitroaromatic chemicals and for downstream biological applications that could utilize pN-Phe as a building block. HighlightsO_LIPara-nitro-L-phenylalanine (pN-Phe) is a valuable small molecule for its applications in genetic code expansion. C_LIO_LIWe establish de novo biosynthesis of pN-Phe from glucose in E. coli, which is also the first example of a de novo pathway design for an unnatural but commonly used non-standard amino acid. C_LIO_LIWe show the first use of an N-oxygenase enzyme in the de novo synthesis of a nitroaromatic product. C_LIO_LIScreening of natural N-oxygenases and strain engineering resulted in final pN-Phe titers of 820 {+/-} 130 {micro}M in shake flask experiments with rich defined media. C_LI