Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α‐hydroxyketones. (10th November 2019)

• Record Type: Journal Article
• Authors: Yu, Haoran; Hernández López, Roberto Icken; Steadman, David; Méndez‐Sánchez, Daniel; Higson, Sally; Cázares‐Körner, Armando; Sheppard, Tom D.; Ward, John M.; Hailes, Helen C.; Dalby, Paul A.
• Is Part Of: FEBS journal. Volume 287:Number 9(2020)
• Page Start: 1758
• Page End: 1776
• Publication Date: 2019-11-10
• Subjects: docking -- protein engineering -- rational design -- transketolase -- two substrates specificity; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.15108
• Languages: English
• ISSNs: 1742-464X

Abstract

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 C2 ‐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. Abstract : Transketolase (TK) from E. coli has been engineered to accept both a novel donor substrate, pyruvate, and unnatural acceptor substrates, including glycolaldehyde, propanal, pentanal, hexanal and 3‐formylbenzoic acid, to produce α‐hydroxyketones. The degree of substrate acceptance of TK has been significantly expanded and the variants obtained have the potential to be used for developing catalytic pathways to produce a diverse range of high‐value products.