초록
<P>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-NO<SUB>2</SUB>-<SMALL>L</SMALL>-tryptophan (Nitrotrp) and its analogues by designing and expressing the biosynthetic pathway in <I>Escherichia coli</I>. The biosynthetic pathway comprised one engineered self-sufficient P450 TB14 of Streptomyces origin for direct nitration of the C-4 of <SMALL>L</SMALL>-Trp indole and one nitric oxide synthase from <I>Bacillus subtilis</I> (BsNOS) for the production of nitric oxide (NO) from <SMALL>L</SMALL>-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 <I>B. subtilis</I> for <I>in situ</I> NADPH regeneration. The initially designed pathway led to 16.2 ± 2.3 mg/L of Nitrotrp by the engineered <I>E. coli</I> 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.</P><BR>[FIG OMISSION]</BR>