Exploiting cell-free systems: Implementation and debugging of a system of biotransformations.
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.
Bujara
Matthias
M
Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
Schümperli
Michael
M
Billerbeck
Sonja
S
Heinemann
Matthias
M
Panke
Sven
S
D001711:Biotransformation; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D009005:Monosaccharides / Q000378:metabolism*
D009005:Monosaccharides; D004099:Dihydroxyacetone Phosphate