Bujara M Schümperli M Billerbeck S Heinemann M Panke S 2010 The 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. application/pdf https://sonar.ch/global/documents/183520 eng info:eu-repo/semantics/altIdentifier/doi/10.1002/bit.22666 info:eu-repo/semantics/altIdentifier/pmid/20091765 info:eu-repo/semantics/openAccess Biotechnology and bioengineering. - 2010 Biotransformation Dihydroxyacetone Phosphate Escherichia coli Metabolic Networks and Pathways Monosaccharides Exploiting cell-free systems: Implementation and debugging of a system of biotransformations. http://purl.org/coar/resource_type/c_6501