Second generation engineering of transketolase for polar aromatic aldehyde substrates

Payongsri, Panwajee; Steadman, David; Hailes, Helen C.; Dalby, Paul A.

doi:10.1016/j.enzmictec.2015.01.008

Cited by 29 publications

(36 citation statements)

References 47 publications

(77 reference statements)

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“…14 A particular class of enzymes known as transketolases (EC 2.2.1.1) have been investigated as biocatalytic tools due to their ability to form the asymmetric carbon-carbon bonds commonly required for chemical synthesis of a broad range of pharmaceuticals, agrochemicals, materials, and food ingredients. 15 In nature transketolases require thiamine diphosphate as a cofactor and provide a regulatory function in cellular metabolism, linking the pentose phosphate pathway to glycolysis by catalyzing transfer of a two-carbon fragment from xylulose to ribose sugars, forming heptulose sugars. Biotechnologists have shown that this activity can be exploited to drive an irreversible reaction when providing bhydroxypyruvate (HPA) as the donor of the two-carbon fragment, resulting in carbon dioxide as one of the reaction products.…”

Section: Introductionmentioning

confidence: 99%

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

Wei

Braun‐Galleani

Henriquez

et al. 2017

Biotechnology Progress

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Transketolase is a proven biocatalytic tool for asymmetric carbon‐carbon bond formation, both as a purified enzyme and within bacterial whole‐cell biocatalysts. The performance of Pichia pastoris as a host for transketolase whole‐cell biocatalysis was investigated using a transketolase‐overexpressing strain to catalyze formation of l‐erythrulose from β‐hydroxypyruvic acid and glycolaldehyde substrates. Pichia pastoris transketolase coding sequence from the locus PAS_chr1‐4_0150 was subcloned downstream of the methanol‐inducible AOX1 promoter in a plasmid for transformation of strain GS115, generating strain TK150. Whole and disrupted TK150 cells from shake flasks achieved 62% and 65% conversion, respectively, under optimal pH and methanol induction conditions. In a 300 μL reaction, TK150 samples from a 1L fed‐batch fermentation achieved a maximum l‐erythrulose space time yield (STY) of 46.58 g L−1 h−1, specific activity of 155 U normalgCDW−1, product yield on substrate (Yp/s) of 0.52 mol mol−1 and product yield on catalyst (Yp/x) of 2.23g normalgCDW−1. We have successfully exploited the rapid growth and high biomass characteristics of Pichia pastoris in whole cell biocatalysis. At high cell density, the engineered TK150 Pichia pastoris strain tolerated high concentrations of substrate and product to achieve high STY of the chiral sugar l‐erythrulose. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:99–106, 2018

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Section: Introductionmentioning

confidence: 99%

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

Wei

Braun‐Galleani

Henriquez

et al. 2017

Biotechnology Progress

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“…In our previous work, variants showing improved performance towards propionaldehyde 5a and HPA also improved the catalytic efficiency towards aromatic substrates with HPA, including 3-FBA 7 or 4-FBA [17,18]. For instance, D469T improved the specific activity towards propionaldehyde 5a and HPA by 5-fold and also improved conversion yield of 3-FBA the new library variants had improved activity towards the aromatic aldehyde 3-FBA 7 and pyruvate 2d (Scheme 3).…”

Section: Novel Enzyme Activity Towards 3-fba and Pyruvatementioning

confidence: 75%

“…Addition of the mutation S385Y into the double mutant D469T/R520Q previously improved the catalytic efficiency towards 3-FBA 7 and HPA by significantly decreasing the Km [18]. We therefore introduced the S385Y mutation into the two mutants 4M/D469T/H473N and 4M/D469T/H473N/R520Q to test whether their activities could be improved.…”

Section: Novel Enzyme Activity Towards 3-fba and Pyruvatementioning

confidence: 99%

“…Wild-type TK accepts a narrow range of hydroxyaldehydes as the aldol-acceptor substrate, and with strict (2R)-specificity [9][10][11]. TK from E. coli has been engineered successfully for improved and reversed enantioselectivity [12], as well as an expanded aldol-acceptor substrate range including polar aliphatic [13], non-polar aliphatic [14], heteroaromatic [15,16] and aromatic aldehyde derivatives [17,18]. Related variants were also introduced by others, into a thermostable TK from Geobacillus stearothermophilus (TKgst) [19][20][21][22].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Simultaneous optimization of donor and acceptor substrate specificity for transketolase by a small but smart library

López

Steadman

et al. 2019

Preprint

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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 optimization of both substrate specificities, is also required for the rapid expansion of accepted substrates. Transketolase catalyses the reversible transfer of a C2ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for widely industrial applications. Herein, transketolase from E. coli was engineered to simultaneously accept both pyruvate as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid. Twenty single-mutant variants were firstly designed and characterized experimentally.Beneficial mutations were then recombined to construct a small but smart library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to WT. Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% ee for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-formylbenzoic acid. 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 acceptances, that 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.

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“…[18,19] In the past ten years many variants of EcTK, GstTK, and Saccharomyces cerevisiae transketolase (ScTK) were createdb y active site targeted directede volution to enhancet he activity for the above mentioned non-phosphorylated substrates. [17,[20][21][22][23][24][25][26][27][28][29][30] Amongt hem also variantsw ith enhanced or reverseds tereoselectivity wered escribed. For details see SI Ta ble S1.…”

Section: Introductionmentioning

confidence: 99%

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

et al. 2018

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A structural model for thiamine‐diphosphate (ThDP)‐dependent transketolase (TK) was developed to analyse the effect of amino acid exchanges on the stereoselectivity of this synthetically important class of enzymes. In this study the carboligation of 3‐hydroxypyruvate as a donor and propanal, as well as pentanal, was studied. Based on literature data and additional mutagenesis studies using E. coli TK, a four‐state model was developed to explain the stereoselectivity of TKs by the relative orientation of donor and acceptor substrates in the active site prior to C−C‐bond formation. To enable a functional comparison of relevant amino acids of TKs from different species, a standard numbering scheme was developed. Using this concept, H26, H261, and F434 were identified as the key residues which mediate stereoselectivity, where two main factors influenced the arrangement of ThDP‐bound donor and acceptor prior to carboligation: the relative orientation of the substrate side chains and the orientation of the acceptor carbonyl group towards the donor hydroxy group. This model provides a first framework to understand the structure‐function relationships of TKs with respect to their stereoselectivity.

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Second generation engineering of transketolase for polar aromatic aldehyde substrates

Cited by 29 publications

References 47 publications

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

Simultaneous optimization of donor and acceptor substrate specificity for transketolase by a small but smart library

Towards a Mechanistic Understanding of Factors Controlling the Stereoselectivity of Transketolase

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