Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan.

Cell-free system has emerged as a powerful platform with a wide range of applications and recently has contributed to express metabolic pathways for biosynthesis. Here we report construction of a native biosynthetic pathway for L-4-nitrotryptophan (L-4-nitro-Trp) synthesis using an -based cell-free protein synthesis (CFPS) system. Naturally, a nitric oxide (NO) synthase (TxtD) and a cytochrome P450 enzyme (TxtE) are responsible for synthesizing L-4-nitro-Trp, which serves as one substrate for the biosynthesis of a nonribosomal peptide herbicide thaxtomin A. Recombinant coexpression of TxtD and TxtE in a heterologous host like for L-4-nitro-Trp production has not been achieved so far due to the poor or insoluble expression of TxtD. Using CFPS, TxtD and TxtE were successfully expressed , enabling the formation of L-4-nitro-Trp. After optimization, the cell-free system was able to synthesize approximately 360 μM L-4-nitro-Trp within 16 h. Overall, this work expands the application scope of CFPS for study and synthesis of nitro-containing compounds, which are important building blocks widely used in pharmaceuticals, agrochemicals, and industrial chemicals.