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

We propose an ecofriendly, efficient, stereoselective procedure for the two-carbon elongation of non-phosphorylated aldoses (C4–C6) to the corresponding Cn+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 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 Cn aldoses as acceptor and HPA as donor substrates were transformed by the TKgst variants at 60°C with practically complete conversion. The corresponding Cn+2 ketoses, including two hexuloses (D-tagatose, L-psicose), two heptuloses (D-altro-heptulose, D-ido-heptulose) and two octuloses ...