Donor Promiscuity of a Thermostable Transketolase by Directed Evolution: Efficient Complementation of 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Activity

Abstract

Enzymes catalyzing asymmetric carboligation reactions typically show very high substrate specificity for their nucleophilic donor substrate components. Structure-guided engineering of the thermostable transketolase from Geobacillus stearothermophilus by directed in vitro evolution yielded new enzyme variants that are able to utilize pyruvate and higher aliphatic homologues as nucleophilic components for acyl transfer instead of the natural polyhydroxylated ketose phosphates or hydroxypyruvate. The single mutant H102T proved the best hit toward 3-methyl-2-oxobutyrate as donor, while the double variant H102L/H474S showed highest catalytic efficiency toward pyruvate as donor. The latter variant was able to complement the auxotrophic deficiency of Escherichia coli cells arising from a deletion of the dxs gene, which encodes for activity of the first committed step into the terpenoid biosynthesis, offering the chance to employ a growth selection test for further enzyme optimization.

nanoDSF as screening tool for enzyme libraries and biotechnology development

“…Transketolase is a dimeric enzyme from a thermophilic bacterium with a rather high T m of 76 °C , but it shows the highest cosolvent sensitivity in our investigation. Its high sensitivity could originate in the tertiary structure where the two active sites are composed of amino acids from both subunits at the dimer interface, where also the organic thiamine cofactor is bound, thereby rendering the enzyme less stable .…”

One‐Pot Cascade Synthesis of (3S)‐Hydroxyketones Catalyzed by Transketolase via Hydroxypyruvate Generated in Situ from d‐Serine by d‐Amino Acid Oxidase

“…On this side, wild-type or evolved TKs from mesophilic [18] or thermophilic [5,6b,19] sources have been reported as efficient biocatalysts for the conversion of a wide range of aldehydes leading to the synthesis of various (3S)-hydroxyketones. In this study, we have selected the mesophilic TK from Escherichia coli (TK eco ) [18d-h] and the thermostable TK from Geobacillus stearothermophilus (TK gst ) [19,20] to be coupled with DAAO Rg in a one-pot two step procedure, starting only from the two substrates d-serine and an appropriated aldehyde acceptor for obtaining valuable (3S)-hydroxyketones.…”

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

“…For biocatalytic applications, mesophilic TKs from Saccharomyces cerevisiae and from Escherichia coli have been largely used and optimized by mutagenesis . We have identified the first thermostable TK from Geobacillus stearothermophilus (TK gst ) that offers significantly improved stability at high temperature, more robustness towards non‐usual reaction conditions and a broadened substrate spectrum obtained by in vitro evolution towards (2 S )‐hydroxylated, aliphatic and aromatic aldehydes and also towards HPA analogs as new donor substrates …”

“…[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).…”