Cell-Free Metabolic Engineering for Butanol Production

Cell-free biological systems have emerged as one approach to decouple the cell’s physiological and evolutionary objectives from the engineer’s process objectives to enable high yielding biomolecular transformations. In this work, we used a crude cell lysate approach to reconstitute the 17-step Clostridium 1-butanol biosynthetic pathway in extracts of Escherichia coli. We prove the ability to mix 5 crude lysates each with selectively over-expressed enzymes to activate the entire 1-butanol production pathway in vitro with high productivities and yields. We additionally demonstrate the ability to quickly optimize these cell-free systems by modulating the physicochemical environment to improve yields and productivities. Furthermore, we establish for the first time the ability to combine cell-free protein synthesis of enzymes with cell-free metabolic engineering to activate the metabolic pathway towards 1-butanol. This work provides a proof-of-concept that not only can we perform chemical synthesis by utilizing cellular metabolism in crude lysates but also that we can construct biosynthetic pathways by using crude lysates for both catalyst synthesis and utilization in one pot. This opens the door for novel biosynthetic pathway prospecting and rapid prototyping of metabolism by cell-free synthetic biology.