Engineering enzyme substrate scope complementarity for promiscuous cascade synthesis

05 September 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Biocatalytic cascades are uniquely powerful for the efficient, asymmetric synthesis of bioactive compounds. The high specificity of enzymes can enable one-pot reactions where the substrates, intermediates, and products react only with the intended enzyme. However, this same specificity can hinder the substrate scope of biocatalytic cascades because each constituent enzyme requires complementary activity. Here, we implement a substrate multiplexed screening (SUMS) approach to improve the substrate scope overlap of a two-enzyme cascade via directed evolution. This cascade leverages an L-threonine transaldolase, ObiH, to produce a range of β-OH amino acids that are subsequently decarboxylated to produce chiral 1,2-amino alcohols. Crucially, for the success of this cascade, we engineered a tryptophan decarboxylase to act efficiently on β-OH amino acids while avoiding activity on L-threonine, which is needed for ObiH activity. We leverage this exquisite selectivity with matched substrate scopes to produce a variety of chiral 1,2-amino alcohols in a one-pot cascade from aldehydes or styrene oxides. This route constitutes a new disconnection for the synthesis of β-adrenergic receptor agonists and shows how SUMS can be used to guide the development of promiscuous, C-C bond forming cascades.


protein engineering
substrate multiplexed screening
directed evolution
pyridoxal phosphate


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