Direct Aromatic Nitration System for Synthesis of Nitrotryptophans in Escherichia coli

Nitrotryptophan and its analogues are useful building blocks for synthesizing bioactive and biotechnologically relevant chemicals, materials, and proteins. However, synthetic routes to enantiopure nitro-containing tryptophan derivatives are either complex and polluting or even unestablished yet. Herein, we describe microbial production of 4-NO₂-l-tryptophan (Nitrotrp) and its analogues by designing and expressing the biosynthetic pathway in Escherichia coli. The biosynthetic pathway comprised one engineered self-sufficient P450 TB14 of Streptomyces origin for direct nitration of the C-4 of l-Trp indole and one nitric oxide synthase from Bacillus subtilis (BsNOS) for the production of nitric oxide (NO) from l-Arg to support the direct aromatic nitration. As both TB14 and BsNOS require reducing agent NADPH for their reactions, we also included one glucose dehydrogenase (GDH) from B. subtilis for in situ NADPH regeneration. The initially designed pathway led to 16.2 ± 2.3 mg/L of Nitrotrp by the engineered E. coli fermented in the M9 minimal medium for 3 days. A combination of the design and screening of three additional pathways, fermentation optimization and the knockout of competitive metabolic pathways together improved the Nitrotrp titer to around 192 mg/L within 20 h. Finally, the whole-cell biotransformation system produced eight Nitrotrp analogues with their titers varying from 2.5 to 61.5 mg/L. This work provides the first microbial direct aromatic nitration processes and sets the stage for the development of biocatalytic routes to other useful nitroaromatics in the future.