Glycerol phosphate oxidase from Streptococcus pneumoniae (GPO<SUB>S.pne</SUB>) was purified and characterized. By the actions of GPO<SUB>S.pne</SUB> and dihydroxyacetone phosphate (DHAP)-dependent aldolases, various ketoses including rare sugars were synthesized with glyceraldehydes as acceptors in a one-pot four-enzyme system.