Evolved Thermostable Transketolase for Stereoselective Two-Carbon Elongation of Non-Phosphorylated Aldoses to Naturally Rare Ketoses

Abstract

We propose an ecofriendly, efficient, stereoselective procedure for the two-carbon elongation of nonphosphorylated aldoses (C4–C6) to the corresponding C n+2 ketoses (C6–C8) in one step, using hydroxypyruvate (HPA) as a ketol donor substrate and an evolved thermostable transketolase from Geobacillus stearothermophilus (TKgst) as a biocatalyst. Simultaneous site saturation mutagenesis (SSM) at two or three key positions in the TKgst active site yielded efficient variants, L382F/F435Y, R521Y/S385/H462N, and R521V/S385D/H462S, with increased activity compared to wild-type TKgst for conversion of two tetroses (d-threose, l-erythrose), two pentoses (d-xylose, d-ribose), and two hexoses (d-allose, d-glucose), respectively. These six C n aldoses as acceptor and HPA as donor substrates were transformed by the TKgst variants at 60 °C with practically complete conversion. The corresponding C n+2 ketoses, including two hexuloses (d-tagatose, l-psicose), two heptuloses (d-altro-heptulose, d-ido-heptulose), and two octuloses (d-glycero- d-ido-octulose, d-glycero-d-altro-octulose) are naturally rare compounds with important biological functions, which were obtained with high diastereoselectivity.

Convergent in situ Generation of Both Transketolase Substrates via Transaminase and Aldolase Reactions for Sequential One‐Pot, Three‐Step Cascade Synthesis of Ketoses

“…Finally, l ‐ribulose 4 , d ‐tagatose 5 and l ‐psicose 6 were isolated in good yields of 53 %, 55 % and 49 %, respectively. It is noteworthy that the isolated yields of compounds 4 – 6 obtained by following the novel cascade process were 4–5 times higher than those obtained with synthetic Li‐HPA and commercially available substrates 1 – 3 , which were reported as 11 %, 13 %, and 10 %, respectively. The three l ‐ erythro (3 S ,4 S ) ketoses were obtained in optically pure form with high diastereoselectivity ( de >95 %).…”

“…[a] Reaction mixtures obtained previously with FSA eco , aldehyde concentrations were quantified by HPLC and quantitative 1 H NMR, [b] Determined using quantitative 1 H NMR relative to 3‐trimethylsilyl‐2,2,3,3‐tetradeuteropropionate (TSPd4) as internal standard [b] Diastereoisomeric excess ( de ) determined by 1 H and 13 C NMR…”

“…These characterization data confirmed that the three TK gst -catalyzed reactions each led to a single ketose with the expected l-erythro (3S,4S) configuration, consistent with the stereoselectivity observed with other aldehyde acceptors by TK gst catalysis at elevated temperatures. [10,12,13]…”

“…For biocatalytic applications, mesophilic TKs from Saccharomyces cerevisiae and from Escherichia coli have been largely used and optimized by mutagenesis. [9,10] We have identified the first thermostable TK from Geobacillus stearothermophilus (TK gst ) that offers significantly improved stability at high temperature, [11] more robustness towards non-usual reaction conditions [12] and a broadened substrate spectrum obtained by in vitro evolution towards (2S)hydroxylated, [13] aliphatic [14] and aromatic aldehydes [15] and also towards HPA analogs as new donor substrates. [16] The purpose of this study was to optimize the synthesis of highly valuable natural (3S,4S)-ketoses from (2S)-hydroxyaldehydes.…”

“…While the synthesis of such compounds is inaccessible using mesophilic TKs, we could recently show that TK gst catalysis enabled best yields upon reaction at 60°C in a reasonable time frame (8-24 h). [11,13] To make this TK gst -catalyzed process greener and more sustainable, a major challenge was to generate the donor HPA as well as the acceptor substrates in situ in a onepot procedure, not only to avoid the costly purchase or chemical synthesis of these compounds, but also to counteract their limited stability at 60°C (Scheme 2). HPA is particularly unstable at high temperature but can be produced in situ from l-serine (L-Ser) by transamination to a ketoacid as amino group acceptor.…”