Retention and biosynthesis of diverse nitroarenes in Escherichia coli after combinatorial nitroreductase gene deletions

The instability of nitroaromatic compounds or nitroarenes in the presence of microbial cells has hindered the ability to biomanufacture these industrially and pharmaceutically relevant chemicals. Although nitroreductase (NTR) gene deletions have been linked to nitroarene stability, the extent of this effect is unclear. Here we perform a comprehensive analysis of bacterial NTR activity in cells by engineering strains of Escherichia coli that contain up to 15 knockouts of known and candidate NTR genes. We evaluate the stability of over 20 exogenously supplemented nitroaromatic compounds to wild-type and engineered strains. For several chemistries, such as di-nitro compounds and nitro-aldehydes, our engineered nitroaromatic reductase knockout strains enable retention of compounds. We leverage these tools and insights to improve or enable nitroaromatic compound biosynthesis, including the biocatalytic transformation of an amine precursor to a nitro compound, the biosynthesis of nitrobenzaldehydes and the combined semi-synthesis and site-specific incorporation of nitrophenylalanines from supplemented nitrobenzaldehydes. This work advances access to nitro functional group chemistry in cell-based biocatalysis, metabolic engineering and synthetic biology. Nitroarenes are unstable in microbial cultures, limiting the biomanufacturing of these industrially and pharmaceutically important chemicals. Genome engineering of nitroreductase-deficient E. coli strains has now been shown to improve nitroarene retention during fermentation, expanding access to nitro functional group chemistry in whole-cell biocatalysis, metabolic engineering and synthetic biology.