Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis.

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

Engineering Enzyme Substrate Scope Complementarity for Promiscuous Cascade Synthesis of 1,2-Amino Alcohols.

Biocatalytic cascades are uniquely powerful for the efficient, asymmetric synthesis of bioactive compounds. However, high substrate specificity can hinder the scope of biocatalytic cascades because the constituent enzymes may have non-complementary activity. In this study, we implemented a substrate multiplexed screening (SUMS) based directed evolution approach to improve the substrate scope overlap between a transaldolase (ObiH) and a decarboxylase for the production of chiral 1,2-amino alcohols. To generate a promiscuous cascade, we engineered a tryptophan decarboxylase to act efficiently on ß-OH amino acids while avoiding activity on l-threonine, which is needed for ObiH activity. We leveraged this exquisite selectivity with matched substrate scope to produce a variety of enantiopure 1,2-amino alcohols in a one-pot cascade from aldehydes or styrene oxides. This demonstration shows how SUMS can be used to guide the development of promiscuous, C-C bond forming cascades.

Modular control of l-tryptophan isotopic substitution via an efficient biosynthetic cascade.

Isotopologs are powerful tools for investigating biological systems. We report a biosynthetic-cascade synthesis of Trp isotopologs starting from indole, glycine, and formaldehyde using the enzymes l-threonine aldolase and an engineered ß-subunit of tryptophan synthase. This modular route to Trp isotopologs is simple and inexpensive, enabling facile access to these compounds.

Facile in Vitro Biocatalytic Production of Diverse Tryptamines.

Tryptamines are a medicinally important class of small molecules that serve as precursors to more complex, clinically used indole alkaloid natural products. Typically, tryptamine analogues are prepared from indoles through multistep synthetic routes. In the natural world, the desirable tryptamine synthon is produced in a single step by l-tryptophan decarboxylases (TDCs). However, no TDCs are known to combine high activity and substrate promiscuity, which might enable a practical biocatalytic route to tryptamine analogues. We have now identified the TDC from Ruminococcus gnavus as the first highly active and promiscuous member of this enzyme family. RgnTDC performs up to 96 000 turnovers and readily accommodates tryptophan analogues with substituents at the 4, 5, 6, and 7 positions, as well as alternative heterocycles, thus enabling the facile biocatalytic synthesis of >20 tryptamine analogues. We demonstrate the utility of this enzyme in a two-step biocatalytic sequence with an engineered tryptophan synthase to afford an efficient, cost-effective route to tryptamines from commercially available indole starting materials.