Using CRISPR/Cas9 for multiplex genome engineering to optimize the ethanol metabolic pathway in Saccharomyces cerevisiae

Despite the great progress in genome editing in the model organism Saccharomyces cerevisiae, regulating the carbon flux to ethanol in the ethanol metabolic pathway to achieve high ethanol yield and productivity remains a challenge. Here, we developed an efficient strategy for single-step, high-efficiency, simultaneous multiple gene disruptions in S. cerevisiae based on the CRISPR/Cas9 system. Three genes, the alcohol dehydrogenase (ADH) 2 gene, the glycerol-3-phosphate dehydrogenase (GPD) 1 gene, and the aldehyde dehydrogenase (ALD) 4 gene, were disrupted singly and combinatorially with efficiency ranging from 80 to 100%. We applied our genome engineering tool to explore all possible single, double, and triple gene disruption combinations to search for strains with high ethanol production. This exploratory analysis identified strains with ethanol production at least 1.41-fold greater than that of the wild-type strain. Our study illustrates the applicability of this highly efficient multiplex genome engineering approach for genome editing and the regulation of metabolic flux in S. cerevisiae.