Abstract
A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding and partly impossible owing to the lack of specific functions or substrates in the host cell, its sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we demonstrate that cell-free systems can be useful in driving the characterization and engineering of biosynthetic pathways. We show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. We prove the concept of this approach on the direct synthesis of indigoidine and rhabdopeptides with the in vitro produced multidomain megasynthases BpsA and KJ12ABC. The in vitro produced proteins are analyzed in detail, i.e., in yield, quality, post-translational modification and specific activity, and compared to recombinantly produced proteins. Our study highlights cell-free PURE systems as suitable setting for the rapid engineering of biosynthetic pathways.