A mutant of D-fructose-6-phosphate aldolase (FSA) of Escherichia coli, FSA A129S, with improved catalytic efficiency towards dihydroxyacetone (DHA), the donor substrate in aldol addition reactions, was explored for synthetic applications. The kcat/KM value for DHA was 17-fold higher with FSA A129S than that with FSA wild type (FSA wt). On the other hand, for hydroxyacetone as donor substrate FSA A129S was found to be 3.5-fold less efficient than FSA wt. Furthermore, FSA A129S also accepted glycolaldehyde (GA) as donor substrate with 3.3-fold lower affinity than FSA wt. This differential selectivity of both FSA wt and FSA A129S for GA makes them complementary biocatalysts allowing a control over donor and acceptor roles, which is particularly useful in carboligation multi-step cascade synthesis of polyhydroxylated complex compounds. Production of the mutant protein was also improved for its convenient use in synthesis. Several carbohydrates and nitrocyclitols were efficiently prepared, demonstrating the versatile potential of FSA A129S as biocatalyst in organic synthesis.