Synthesis of (R,R)-2,3-butanediol from starch in a hybrid cell-free reaction system

In this study, we demonstrate the conversion of starch to (R,R)-2,3-butanediol (2,3-BD) in a hybrid cell-free synthesis system containing a mixture of lysates derived from Escherichia coli (E. coli) and cyanobacteria. A sufficient pool of pyruvate required for the synthesis of 2,3-BD was generated by combining metabolic pathways of cyanobacteria and E. coli. Successful synthesis of 2,3-BD was achieved by additional modifications of the hybrid cell-free system with the enzymes required to convert pyruvate to 2,3-BD. The results demonstrate a new approach to harness biological pathways to expand the scope of cell-free metabolic engineering by cross-species combinations of cell lysates.