| Bioethanol,as a form of renewable and clean energy,has been recognized as the most promising alternative to fossil fuels.Saccharomyces cerevisiae plays an important role in bioethanol production due to its high ethanol tolerance,clear genetic background and ability to ferment a wide range of sugars.In this study,we applied CRISPR/Cas9 system to manipulate the relevant genes in the metabolic pathways of ethanol,glycerin and acetic acid in the anaerobic fermentation process of S.cerevisiae.By regulating the metabolic flow to reduce the by-products,thereby improving the conversion rate of ethanol.We firstly selected alcohol dehydrogenases II(ADH2)genes as target,and used CRISPR/Cas9 technology for gene knockout.Sanger sequencing showed the accurate knockout of the target gene with efficiency ranging from 70 to 100%.Subsequently,we utilized genome resequencing to validate the mutations in the ADH2 mutants targeted by various single guide RNAs.Paired?end alignment and SNP calling showed that these mutations were consistent with the theoretical design.The ethanol fermentation experiment showed that the ethanol yield improved by up to 74.7% compared with the yield obtained using the native strain.By constructing multiple sgRNA expression cassette vectors for multi-locus gene editing,we selected ADH2 gene,glycerol-3-phosphate dehydrogenase(GPD1)gene and aldehyde dehydrogenase(ALD4)gene as target genes for genome engineering of up to three different genomic loci in one transformation step in S.cerevisiae.Sequencing of mutant strains demonstrated the interruption of the target gene with efficiency ranging from 75 to 100%.Subsequently,ethanol fermentation experiments were carried out with single,double and triple gene disrupted mutants.The results showed that the ethanol yield of YS-7 was increased by 22.54%,and the yield of glycerol and acetic acid was decreased by 36.11% and 25.93%,respectively.Antisense RNA interference technology was applied to interfere with the expression of GPD1 gene.While interrupting the ADH2 gene,the target site was knocked into the antisense interference component of the GPD1 gene(PGK-SGPD1-CYC1).The CRISPR/Cas9 system mediated recombination efficiencies of 43.40 % were achieved.Fermentation test showed that the ethanol yield of the mutant strain SG1-1 was 9.07% higher than the wild type,and the yield of glycerol and acetate were decreased by 12.05% and 12.30%,respectively. |
