Design Space Exploration of the Violacein Pathway in Escherichia coli Based Cell-Free System

In this study, an Escherichia coli (E. coli) based transcription translation cell-free system (TX-TL) was employed to sample various enzyme expression levels of the violacein pathway. The pathway was successfully reconstructed in TX-TL. Its variations produced different metabolites as evident from the extracts’ assorted colors. Analysis of the violacein product via UV-Vis absorption and liquid chromatography-mass spectrometry (LC-MS) detected 68 ng of violacein per 10 µl reaction volume. Significant buildup of prodeoxyviolacein intermediate was also detected in the equimolar TX-TL reaction. Finally, design space exploration experiments suggested an improvement in violacein production at high VioC and VioD DNA concentrations.