Transketolase (TK) was used as biocatalyst to produce useful aldoses that serve as aldolase electrophiles to prepare monosaccharides of biological interest. Unconventionally, TK was implemented in a synthetic pathway, as in metabolism, i.e., without using a specially designed nucleophile such as hydroxypyruvate to shift the equilibrium. Unusually, formaldehyde was taken as the general TK acceptor substrate to generate aldehydes. Here it was demonstrated that this method can be generalized: the substrates and the aldolase were successfully varied, coupled with other enzymes in a one-pot, one-step green cascade reaction process involving four enzymes with atom economy. In addition, new assays were developed for both monitoring the progress of the reactions and assessing the purity of the synthesized products.