AbstractThe orchestration of a multitude of enzyme catalysts allows cells to carry out complex and thermodynamically unfavorable chemical conversions. In an effort to recruit these advantages for in vitro biotransformations, we have assembled a 10‐step catalytic system—a system of biotransformations (SBT)—for the synthesis of unnatural monosaccharides based on the versatile building block dihydroxyacetone phosphate (DHAP). To facilitate the assembly of such a network, we have insulated a production pathway from Escherichia coli's central carbon metabolism. This pathway consists of the endogenous glycolysis without triose‐phosphate isomerase to enable accumulation of DHAP and was completed with lactate dehydrogenase to regenerate NAD+. It could be readily extended for the synthesis of unnatural sugar molecules, such as the unnatural monosaccharide phosphate 5,6,7‐trideoxy‐D‐threo‐heptulose‐1‐phosphate from DHAP and butanal. Insulation required in particular inactivation of the amn gene encoding the AMP nucleosidase, which otherwise led to glucose‐independent DHAP production from adenosine phosphates. The work demonstrates that a sufficiently insulated in vitro multi‐step enzymatic system can be readily assembled from central carbon metabolism pathways. Biotechnol. Bioeng. 2010; 106: 376–389. © 2010 Wiley Periodicals, Inc.