Engineering a Thermostable Transketolase for Unnatural Conversion of (2<i>S</i>)‐Hydroxyaldehydes

Abstract

Abstract Transketolase (TK) from various origins (including Escherichia coli and yeast) has been described to be fully enantiomer specific for (2 R )‐hydroxyaldehyde substrates. A thermostable TK from G eobacillus stearothermophilus (TK gst ) was found to display a minor reactivity for (2 S )‐hydroxylated aldehydes. To improve this activity by directed protein evolution, we have built a library of TK gst variants by site saturation mutagenesis on two key positions L382 and D470. The best TK gst double mutant L382D/D470S shows up to 4‐ and 5‐fold higher activities towards L ‐lactaldehyde and L ‐glyceraldehyde as acceptor substrates, respectively. Preparative utility of this mutant was demonstrated by the one‐step synthesis of valuable L ‐ribulose and its 5‐deoxy analogue with the L ‐ erythro (3 S ,4 S ) configuration, which were previously inaccessible by using common TK sources. magnified image