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 μM 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.

Challenges and opportunities in bringing nonbiological atoms to life with synthetic metabolism

“…Notably, iridium and rhodium (as replacement for Fe in myoglobin and P450 enzymes) catalyze C-H shifts, cyclopropanation of inactivated alkenes, and intramolecular nitrene C-H insertions, chemical reactions unknown to biology [139][140][141][142]. Although there are no known examples of novel cofactorcatalyzed biochemical reactions for introducing nonbiological atoms into the core biochemistry of microbial hosts, exploiting chemical characteristics of elements to tailor desired enzyme functionalities is an appealing feature to be explored [143]. A recent publication described an additional technique for inserting non-canonical atoms into biological systems.…”