From de novo Enzymes to Synthetic Metabolic Pathways Synthetic Biology for Industrial BiotechnologyOur ability to design cell factories to produce valuable chemicals requires the "recombination" not only of existing but also designer enzymes into novel metabolic pathways to achieve entirely new metabolic function. This poses two distinct challenges that need to be solved in an integrated way if we ambition to fully deliver on the promise of synthetic biology. The first challenge deals with the rapid design of novel enzymes with high level of activities for reactions not known to be catalyzed in nature. To this end, Arzeda is developing high-throughput computational methodologies to exploit natural enzyme latent catalytic promiscuity to rapidly design new catalysts de novo. This will be illustrated with a couple of successful industrial examples. The second challenge is that of finding optimal ways to arrange natural and designed enzymes to biosynthetize a small-molecule of interest, effectively creating a synthetic metabolism. To illustrate our progress towards solving this challenge, we will discuss Scylax?, a novel tool that Arzeda has been developing for the automated design of novel biosynthetic pathways. Inspired by retrosynthetic methods in organic chemistry, our software draws on databases of known natural enzymatic reactions as well as reactions that can be catalyzed by computationally designed enzymes to exhaustively enumerate biosynthetic routes from a set of desired metabolites down to any small molecule of interest. Pathways are ranked based on thermodynamic feasibility and designability of each enzymatic step. Designed pathways are then validated in-vitro and the best ones introduced in fermentation chassis (prototyping) and further improved using traditional metabolic engineering techniques.