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 nonphosphorylated 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 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 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 (d-glycero-d-ido-octulose, d-glycero-d-altro-octulose) are naturally rare compounds
with important biological functions, which were obtained with high
diastereoselectivity.