LAUSR

The hydroxylation of tryptophan is an important reaction in the biosynthesis of natural products. 5‐Hydroxytryptophan (5HTP) is not only an important compound for its pharmaceutical value but also because it is the precursor of other molecules, such as serotonin. In this study, we have extended the metabolism of an E. coli strain to produce 5HTP. Aromatic amino acid hydroxylase from Cupriavidus taiwanensis (CtAAAH) was selected using an in silico structure‐based approach. We have predicted and selected several substrate‐determining residues using sequence, phylogenetic and functional divergence analyses; we also did rational design on CtAAAH to shift the enzyme preference from phenylalanine to tryptophan. Whole cell bioconversion assays were used to show the effect of predicted sites. In general, all of them decreased the preference toward phenylalanine and increased the tryptophan synthesis activity. The best performer, CtAAAH‐W192F, was transformed into a strain that had the tryptophanase gene disrupted and carried a human tetrahydrobiopterin (BH4) regeneration pathway. The resulting strain was capable of synthesizing 2.5 mM 5HTP after 24 hours. This work demonstrates the application of computational approaches for protein engineering and further coupling with the bacterial metabolism.