Assembly of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative plasmid toolbox

A robust and versatile tool for multigene pathway assembly is a key to the biosynthesis of high-value chemicals. Here we report the rapid construction of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative toolbox (pUMRI) developed in our research group, which has features of ready-to-use, convenient marker recycling, arbitrary element replacement, shuttle plasmid, auxotrophic marker independence, GAL regulation, and decentralized assembly. Functional isoprenoid biosynthesis pathways containing 4-11 genes with lengths ranging from similar to 10 to similar to 22 kb were assembled using this toolbox within 1-5 rounds of reiterative recombination. In combination with GAL-regulated metabolic engineering, high production of isoprenoids (e.g., 16.3 mga (TM) g(-1) dcw carotenoids) was achieved. These results demonstrate the wide range of application and the efficiency of the pUMRI toolbox in multigene pathway construction of S. cerevisiae.