Development and optimization of an engineered E. coli platform for nitrotryptophan biosynthesis.

Nitrotryptophan and its derivatives are valuable building blocks for synthesizing bioactive compounds and functional materials. This study reports the development of an efficient and novel bio-catalytic bioreactor in Escherichia coli capable of the direct aromatic nitration of tryptophan, enabling the synthesis of nitrotryptophan isomers. The biosynthetic pathway incorporates a self-sufficient P450 enzyme (TB14, consisting of TxtE-linker14-BM3R) from Streptomyces for the direct insertion of a nitro group into the indole ring of L-tryptophan. This process is supported by a nitric oxide synthetase (BsNOS) from Bacillus subtilis or its chemical alternative, sodium nitroprusside (SNP), to produce nitric oxide (NO) from L-arginine, which facilitates the direct nitration. As both TB14 and BsNOS require the reductant NADPH for their respective biochemical reactions, a glucose dehydrogenase (GDH) from Bacillus subtilis was included in the experimental design to ensure NADPH regeneration within the system.The initial engineered strain produced 133.2 mg/L of nitrotryptophan in TB medium. Through systematic optimization, including pathway balancing, fermentation condition enhancement, and elimination of competing metabolic pathways, the final titer was successfully increased to 209.9 mg/L within 48 h. This work establishes a robust platform for the microbial production of valuable nitroaromatic compounds and provides key insights for future biocatalytic nitration strategies.