From drug scaffolds to chemical probes, L-tryptophan plays key roles in biological systems and is increasingly valued in pharmaceutical and synthetic biology applications [1,2]. While tryptophan synthase (TrpS) achieves near-stoichiometric indole : serine ratios, it often requires protein engineering to broaden its substrate scope to non-canonical substrates [3]. E. coli  Tryptophanase (TnaA), known since the work of Snell (1964) to catalyze L-tryptophan synthesis from indole and serine, offers a wider substrate tolerance [4], but high excess of serine is required. However, L-serine is not the only option, and alternative nucleophiles may offer a better performance. Through a systematic study of the electrophilic component of the reaction, we have identified S-Benzyl-L-Cysteine (SBC) which reduces the required equivalents by 6 fold (to 1:2 ratio), accelerates the reaction rate and enables straightforward product isolation, by exploiting the low solubility of the benzylmercaptan byproduct in the buffer (Figure 1).  

Figure 1. Biosynthesis of L-tryptophan from Indole and S-Benzyl-L-Cysteine. 

Combined with an extensive nucleophilic substrate scope across indole derivatives and promising scalability, this work estabilishes TnaA as a versatile platform for the sustainable production of tryptophan-based pharmaceutical building blocks, offering a competitive alternative to canonical tryptophan synthase systems [5]. 

[1] D. K. Romney, J. Murciano-Calles, J. E. Wehrmüller and F. H. Arnold, J. Am. Chem. Soc., 2017, 139, 10769–10776. 
[2] S. Xiao, Z. Wang, B. Wang, B. Hou, J. Cheng, T. Bai, Y. Zhang, W. Wang, L. Yan and J. Zhang, Front. Microbiol., 2023, 14, 1099098. 
[3] A. R. Buller, S. Brinkmann-Chen, D. K. Romney, M. Herger, J. Murciano-Calles and F. H. Arnold, Proc. Natl. Acad. Sci. U. S. A., 2015, 112, 14599–14604. 
[4] W. A. Newton and E. E. Snell, Proc. Natl. Acad. Sci. United States, 1964, 51, 382–389. 
[5] E. Sgroi, F. Paradisi, in preparation