Current methods for assembling metabolic pathways require a process of repeated trial and error and have a long design-build-test cycle. Further, it remains a challenge to precisely tune enzyme expression levels for maximizing target metabolite production. Recently it was shown that a cell-free transcriptional-translation system (TX-TL) can be used to rapidly prototype novel complex biocircuits as well as metabolic pathways. TX-TL systems allow protein expression from multiple DNA pieces, opening up the possibility of modulating concentrations of DNA encoding individual pathway enzymes and testing the related effect on metabolite production. In this work, we demonstrate TX-TL as a platform for exploring the design space of metabolic pathways using a 1,4-BDO biosynthesis pathway as an example. Using TX-TL, we verified enzyme expression and enzyme activity and identified the conversion of 4-hydroxybutyrate to downstream metabolites as a limiting step of the 1,4-BDO pathway. We further tested combinations of various enzyme expression levels and found increasing downstream enzyme expression levels improved 1,4-BDO production.

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