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

Abstract

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 β‐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.

Multiplexed Assessment of Promiscuous Non-Canonical Amino Acid Synthase Activity in a Pyridoxal Phosphate-Dependent Protein Family

“…This substrate multiplexed screening (SUMS) approach offers several distinct advantages compared to single-substrate measurements. 61,62 In SUMS unimolecular transformations, the ratio of the products is directly proportional to the ratio of the catalytic efficiencies. For bimolecular reactions, however, complex kinetic phenomena can break the pseudo-first-order relationship between catalytic efficiency and specificity.…”

Multiplexed deconvolution of enzyme function in a PLP-dependent protein family

“…This substrate multiplexed screening (SUMS) approach offers several distinct advantages compared to single substrate measurements. 34,35 Comparison of relative rates of product formation from single-substrate reactions provides only indirect information about specificity. By assaying multiple substrates in competition, SUMS gives direct information on the ability of an enzyme to discriminate between two or more substrates.…”

Styrene Oxide Isomerase‐Catalyzed Meinwald Rearrangement in Cascade Biotransformations: Synthesis of Chiral and/or Natural Chemicals

“…Two examples of epoxides 1 were also converted via this sequence of isomerization, CÀ C bond formation and decarboxylation to produce enantiopure benzylic 1,2-amino alcohols 22 a and 22 b in 27 to 33 % isolated yields (Scheme 5b). [37] The same group also demonstrated that aldehydes 2 generated in situ by SOI from 1 can be intercepted by another enzyme that catalyzes a decarboxylative aldol addition (UstD v2.0 ), thus producing γ-hydroxy amino acids 23 a and 23 b, albeit with low diastereoselectivity of 5 : 2 to 7 : 2 (Scheme 5c). [34] Cascades Involving SOI-Catalyzed Epoxide Isomerization to Synthesize Natural Chemicals from Renewable Substrates SOI has been incorporated as a key enzyme in cascade biotransformations to convert renewable bio-based substrates into high-value products.…”

“…Racemic 6 a was converted by 0.1 %w/v whole cells harbouring SOI to generate 4 a in situ, which was subsequently converted by purified ObiH and Rgn TDC NMY to produce benzylic 1,2‐amino alcohol 21 a as a single diastereomer in 61 % analytical yield and 39 % isolated yield. Two examples of epoxides 1 were also converted via this sequence of isomerization, C−C bond formation and decarboxylation to produce enantiopure benzylic 1,2‐amino alcohols 22 a and 22 b in 27 to 33 % isolated yields (Scheme 5b) [37] . The same group also demonstrated that aldehydes 2 generated in situ by SOI from 1 can be intercepted by another enzyme that catalyzes a decarboxylative aldol addition (UstD v2.0 ), thus producing γ ‐hydroxy amino acids 23 a and 23 b , albeit with low diastereoselectivity of 5 : 2 to 7 : 2 (Scheme 5c) [34] …”