Large-scale pathway reconstruction and colorimetric screening accelerate cellular metabolism engineering

The capability to manipulate and analyze hard-wired metabolic pathways sets the pace at which we can engineer cellular metabolism. Here, we present a framework to extensively rewrite the central metabolic pathway for malonyl-CoA biosynthesis in yeast and readily assess malonyl-CoA output based on pathway-scale DNA reconstruction in combination with colorimetric screening (Pracs). We applied Pracs to generate and test millions of enzyme variants by introducing genetic mutations into the whole set of genes encoding the malonyl-CoA biosynthetic pathway and identified hundreds of beneficial enzyme mutants with increased malonyl-CoA output. Furthermore, the synthetic pathways reconstructed by randomly integrating these beneficial enzyme variants generated vast phenotypic diversity, with some displaying higher production of malonyl-CoA as well as other metabolites, such as carotenoids and betaxanthin, thus demonstrating the generic utility of Pracs to efficiently orchestrate central metabolism to optimize the production of different chemicals in various metabolic pathways. Pracs will be broadly useful to advance our ability to understand and engineer cellular metabolism.