초록
<P><B>Abstract</B></P> <P>Despite the great progress in genome editing in the model organism <I>Saccharomyces cerevisiae</I>, 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 <I>S. cerevisiae</I> 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 <I>S. cerevisiae</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Multiplex CRISPR/Cas9 system has been developed for 1–3 genome edits in <I>S. cerevisiae</I>. </LI> <LI> High efficiencies of multi-gene disruptions obtained vary from 80% to 100%. </LI> <LI> Combinatorial effect on ethanol level identified from multiple knockout were observed. </LI> <LI> Ethanol productivity of the strain increased 1.41-fold compared to native strain. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>