Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α‐hydroxyketones

Abstract

A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimisation of both substrate specificities is also required for the rapid expansion of accepted substrates. Transketolase (TK) catalyses the reversible transfer of a C 2 -ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for industrial applications. Herein, TK from Escherichia coli was engineered to accept both pyruvate, as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid (FBA). Twenty single-mutant variants were first designed and characterised experimentally. Beneficial mutations were then recombined to construct a small library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to wild-type (WT). Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% enantiomeric excess (ee) for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-FBA. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor recognition, which would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high-value products.

Engineering of enzymes using non-natural amino acids

“…Similar results were obtained when a histidine in the substrate binding pocket of TK was replaced by 3MH. By analysing the crystal structures of TK complexed with donor substrates, the previous studies showed H100 of E. coli and yeast TK forms hydrogen bonds with the C1-hydroxy groups of its donor substrates including D-xylulose-5-phosphate (X5P) and D-fructose-6-phosphate (F6P) [ 38–40 ], and so switching the substrate to pyruvate would remove this hydrogen bond due to the lack of the hydroxyl group. Thus, we conjectured that by adding a methyl group to H100, its hydrophobic interaction with pyruvate may be enhanced, thus allowing the substrate to be anchored.…”

Application Fields, Positions, and Bioinformatic Mining of Non-active Sites: A Mini-Review

“…For example, in order to engineer transketolase (TK) to accept an unnatural donor (pyruvate) and acceptor (aliphatic or aromatic aldehydes), Yu et alperformedstructural alignment between TK and pyruvate decarboxylase (PDC). Furthermore, amino acid residues of TK, those around the substrate of PDC, were selected for mutagenesis (Yu et al, 2020). Thus, non-active sites could be considered good candidates for improving enzymeperformance.…”

Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis

“…This forms a new asymmetric α-hydroxyketone with an (S)-configuration, which is otherwise difficult to prepare chemically. 6,7 WT and engineered TKs from different microbial sources have been used in the synthesis of phosphorylated sugars, 8 non-phosphorylated aliphatic and aromatic acyloins, [9][10][11][12][13] and rare sugars such as L-glucoheptulose, 14,15 as well as in coupled cascade reactions. [15][16][17][18] For synthetic applications, biocatalysis using enzymes such as TK allows a sustainable, one-step stereoselective way for preparing chiral building blocks and fine chemicals.…”