A novel cascade reaction for the production of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose was studied in this work. The strategy combines three enzymes in one pot: (i) horse liver alcohol dehydrogenase for the oxidation of N-Cbz-3-aminopropanol to the corresponding aldehyde, (ii) NADH oxidase for the regeneration of coenzyme NAD⁺ and (iii) D-fructose-6-phosphate aldolase from E. coli A129S variant for the aldol addition of dihydroxyacetone to N-Cbz-3-aminopropanal. On the basis of preliminary experiments, optimization of the initial reaction conditions was done using statistical methods, i.e. factorial design of experiments. 79% yield of aldol adduct was achieved in the batch reactor after optimization.