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

Abstract

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 ~10 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 mg∙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.

Graphical abstract

Keywords

pathway assembly / toolbox / reiterative recombination / S. cerevisiae / biosynthesis

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Higher Education Press and Springer-Verlag Berlin Heidelberg