Tryptophanase Mining and Characterization toward the Biological Production of Indole Derivatives.

Tryptophan is an essential amino acid, and its fermentation-based synthesis is well-described with double-digit titers. Therefore, tryptophanase may serve as the gateway enzyme toward indole-derived biobased molecules. They are widespread PLP-dependent enzymes with a conserved active site. Even though their function and structure are well described, the number of characterized tryptophanases is rather low. Here, we characterized a panel of tryptophanases mined from public databases, aimed to explore their industrial amenability. We used the machine learning method variational autoencoding to visualize the tryptophanase sequence space and ensure that the natural sequence diversity is covered in our panel. After initial experiments, the gathered information was used to identify 2 additional regions of interest in the visualized latent space. The solubly expressed enzymes (18 of 21) were biochemically characterized for optimal reaction conditions and kinetic parameters. The characterized panel encompassed a diverse set of tryptophanases with distinct optimal conditions, facilitating the selection of an appropriate candidate. The most suitable tryptophanase, PreTIL, was used in an enzymatic cascade toward biobased indigo synthesis. Optimal cosubstrate and enzyme concentrations were determined by Design of Experiments, resulting in 3.0 ± 0.8 mM indigo formation in situ. Though this titer is comparable to reports on indigo synthesis through fermentation, a fundamental phenomenon appears to halt biocatalytic indigo synthesis.