In Vitro Reconstitution and Optimization of the Entire Pathway to Convert Glucose into Fatty Acid

Glucose and fatty acids play essential physiological roles in nearly all living organisms, and the pathway that converts glucose into fatty acid is pivotal to the central metabolic network. We have successfully reconstituted a pathway that converts glucose to fatty acid in vitro using 30 purified proteins. Through systematic titration and optimization of the glycolytic pathway and pyruvate dehydrogenase, we increased the yield of free fatty acid from nondetectable to a level that exceeded 9% of the theoretical yield. We also reconstituted the entire pentose-phosphate pathway of Escherichia coli and established a pentose phosphate-glycolysis hybrid pathway, replacing GAPDH to enhance NADPH availability. Our efforts provide a useful platform for research involving these core biochemical transformations.