Nitrene generation and transfer for unnatural biosynthesis in living cells

Donnell, Isaac; Quest, Andrew (ORCID:0009000450813664); Tang, Jeremy (ORCID:0009000391529777); Zhang, Maodun; Bloomer, Brandon; Clark, Douglas S. (ORCID:000000031516035X); Mukhopadhyay, Aindrila (ORCID:0000000265137425); Hartwig, John (ORCID:000000024157468X); Keasling, Jay D. (ORCID:0000000341706088)

doi:10.1038/s41557-026-02224-4

DOE OSTI · 3685154

Nitrene generation and transfer for unnatural biosynthesis in living cells

Donnell, Isaac·Quest, Andrew (ORCID:0009000450813664)·Tang, Jeremy (ORCID:0009000391529777)·Zhang, Maodun·Bloomer, Brandon·Clark, Douglas S. (ORCID:000000031516035X)·Mukhopadhyay, Aindrila (ORCID:0000000265137425)·Hartwig, John (ORCID:000000024157468X)·Keasling, Jay D. (ORCID:0000000341706088)

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Abstract

Abstract Synthetic biology has enabled the production of natural and unnatural products from inexpensive sustainable feedstocks. Yet, the scope of available products has been limited largely to compounds accessible from nature’s chemical reactions. Evolved enzymes can catalyse reactions of unnatural substrates or reactions not found in nature but often require the addition of synthetic reagents to purified enzymes or to resting cells containing those enzymes. Here we show that chemical reactions of metabolic intermediates produced intracellularly in living cells can include intermolecular nitrene transfers. The biosynthesis of N -acetoxyanilines, in combination with the generation and transfer of N -aryl nitrene intermediates from them catalysed by a cytochrome P450, generates amino alcohols, diamines, diarylamines and aminoalkyl arenes from simple carbon feedstocks. These products—common substructures of pharmaceuticals and agrochemicals—are challenging to synthesize by standard organic chemistry and were produced from inexpensive, renewable feedstocks. Evolution of the enzymes in this pathway showed that titres can be increased by engineering and that the products can be synthesized with high enantioselectivity.

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Donnell, Isaac, Quest, Andrew (ORCID:0009000450813664), Tang, Jeremy (ORCID:0009000391529777), Zhang, Maodun, Bloomer, Brandon, Clark, Douglas S. (ORCID:000000031516035X), Mukhopadhyay, Aindrila (ORCID:0000000265137425), Hartwig, John (ORCID:000000024157468X), Keasling, Jay D. (ORCID:0000000341706088). 2026-08-18. Nitrene generation and transfer for unnatural biosynthesis in living cells. https://doi.org/10.1038/s41557-026-02224-4

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