Improved bioethanol production using CRISPR/Cas9 to disrupt the ADH2 gene in Saccharomyces cerevisiae

Xue, Ting; Liu, Kui; Chen, Duo; Xue, Yuan; Fang, Jingping; Yan, Hansong; Huang, Luqiang; Chen, Youqiang; He, Wei

doi:10.1007/s11274-018-2518-4

Cited by 31 publications

(14 citation statements)

References 46 publications

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“…Therefore, manipulating certain oxidation-reduction processes might improve the efficiency of ethanol production. For example, overexpression of NADH oxidase to maintain the redox balance [57], deletion of ADH that involved in ethanol utilization [58] or deletion of mitochondrial NADH dehydrogenases to shift from respiratory to fermentative metabolism [59]. Further research is required to test these ideas experimentally.…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering of the cellulolytic thermophilic fungus Myceliophthora thermophila to produce ethanol from cellobiose

Zhang

et al. 2020

Biotechnol Biofuels

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Background: Cellulosic biomass is a promising resource for bioethanol production. However, various sugars in plant biomass hydrolysates including cellodextrins, cellobiose, glucose, xylose, and arabinose, are poorly fermented by microbes. The commonly used ethanol-producing microbe Saccharomyces cerevisiae can usually only utilize glucose, although metabolically engineered strains that utilize xylose have been developed. Direct fermentation of cellobiose could avoid glucose repression during biomass fermentation, but applications of an engineered cellobiose-utilizing S. cerevisiae are still limited because of its long lag phase. Bioethanol production from biomass-derived sugars by a cellulolytic filamentous fungus would have many advantages for the biorefinery industry. Results: We selected Myceliophthora thermophila, a cellulolytic thermophilic filamentous fungus for metabolic engineering to produce ethanol from glucose and cellobiose. Ethanol production was increased by 57% from glucose but not cellobiose after introduction of ScADH1 into the wild-type (WT) strain. Further overexpression of a glucose transporter GLT-1 or the cellodextrin transport system (CDT-1/CDT-2) from N. crassa increased ethanol production by 131% from glucose or by 200% from cellobiose, respectively. Transcriptomic analysis of the engineered cellobioseutilizing strain and WT when grown on cellobiose showed that genes involved in oxidation-reduction reactions and the stress response were downregulated, whereas those involved in protein biosynthesis were upregulated in this effective ethanol production strain. Turning down the expression of pyc gene results the final engineered strain with the ethanol production was further increased by 23%, reaching up to 11.3 g/L on cellobiose. Conclusions: This is the first attempt to engineer the cellulolytic fungus M. thermophila to produce bioethanol from biomass-derived sugars such as glucose and cellobiose. The ethanol production can be improved about 4 times up to 11 grams per liter on cellobiose after a couple of genetic engineering. These results show that M. thermophila is a promising platform for bioethanol production from cellulosic materials in the future.

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Section: Discussionmentioning

confidence: 99%

Metabolic engineering of the cellulolytic thermophilic fungus Myceliophthora thermophila to produce ethanol from cellobiose

Zhang

et al. 2020

Biotechnol Biofuels

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“…GPD2 and FPS1 deletion in S. cerevisiae caused the decrease of glycerol content by 7.95 and 18.8%, respectively [ 32 , 33 ]. ADH2 deletion in S. cerevisiae also resulted in the improvement of ethanol yield [ 34 ]. This study showed that the growth rate of ethanol yield in SCGFA with GPD2 , FPS1 , and ADH2 deletion was higher than SCGF with double-deletion GPD2 and FPS1 , SCG with single-deletion GPD2 , and SCF with single-deletion FPS1 .…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 Approach Constructed Engineered Saccharomyces cerevisiae with the Deletion of GPD2, FPS1, and ADH2 to Enhance the Production of Ethanol

Yang

Jiang

et al. 2022

JoF

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Bioethanol plays an important value in renewable liquid fuel. The excessive accumulation of glycerol and organic acids caused the decrease of ethanol content in the process of industrial ethanol production. In this study, the CRISPR-Cas9 approach was used to construct S. cerevisiae engineering strains by the deletion of GPD2, FPS1, and ADH2 for the improvement of ethanol production. RNA sequencing and transcriptome analysis were used to investigate the effect of gene deletion on gene expression. The results indicated that engineered S. cerevisiae SCGFA by the simultaneous deletion of GPD2, FPS1, and ADH2 produced 23.1 g/L ethanol, which increased by 0.18% in comparison with the wild-type strain with 50 g/L of glucose as substrate. SCGFA strain exhibited the ethanol conversion rate of 0.462 g per g of glucose. In addition, the contents of glycerol, lactic acid, acetic acid, and succinic acid in SCGFA decreased by 22.7, 12.7, 8.1, 19.9, and 20.7% compared with the wild-type strain, respectively. The up-regulated gene enrichment showed glycolysis, fatty acid, and carbon metabolism could affect the ethanol production of SCGFA according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Therefore, the engineering strain SCGFA had great potential in the production of bioethanol.

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“…GPD2 and FPS1 deletion in S. cerevisiae caused the decrease of glycerol content by 7.95 and 18.8%, respectively [25,26]. ADH2 deletion in S. cerevisiae also resulted in the improvement of ethanol yield [27]. This study showed that the growth rate of ethanol yield in SCGFA with GPD2, FPS1, and ADH2 deletion was higher than SCGF with double-deletion GPD2 and FPS1, SCG with single-deletion GPD2, and SCF with single-deletion FPS1.…”

Section: Discussionmentioning

confidence: 99%

Improvement of the Bioethanol Yield in Saccharomyces cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta Constructed by the CRISPR-Cas9 Approach with the Decrease of By-Product Formation

Yang

Jiang

et al. 2022

Preprint

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Background: Bioethanol plays an important value in renewable liquid fuel. The inhibition of by-product formation would enhance the ethanol production of Saccharomyces cerevisiae. In fact, the excessive accumulation of glycerol and organic acids caused the decrease of ethanol content in the process of industrial ethanol production. Results: In this study, S. cerevisiae engineering strains were constructed using the CRISPR-Cas9 approach to delete GPD2, FPS1, and ADH2 to improve the yield of ethanol with the decrease of by-product contents. Engineered S. cerevisiae SCGFA by the GPD2, FPS1, and ADH2 deletion produced 23.1 g/L with 50 g/L of glucose as substrate. SCGFA strain exhibited the ethanol conversion rate of 0.462 g per g of glucose. In addition, the contents of glycerol, lactic acid, acetic acid, and succinic acid in SCGFA decreased by 22.7, 12.7, 8.1, 19.9, and 20.7% compared with the wild-type strain, respectively. Conclusions: The co-knockout of GPD2, FPS1, and ADH2 dramatically improved the ethanol yield of S. cerevisiae by the inhibition of glycerol release and the prevention of ethanol consumption. This study developed a new S. cerevisiae engineering strain SCGFA with high-level ethanol production after GPD2, FPS1, and ADH2 deletion. The engineering strain SCGFA could apply in ethanol production with less formation of by-products.

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Improved bioethanol production using CRISPR/Cas9 to disrupt the ADH2 gene in Saccharomyces cerevisiae

Cited by 31 publications

References 46 publications

Metabolic engineering of the cellulolytic thermophilic fungus Myceliophthora thermophila to produce ethanol from cellobiose

Metabolic engineering of the cellulolytic thermophilic fungus Myceliophthora thermophila to produce ethanol from cellobiose

CRISPR-Cas9 Approach Constructed Engineered Saccharomyces cerevisiae with the Deletion of GPD2, FPS1, and ADH2 to Enhance the Production of Ethanol

Improvement of the Bioethanol Yield in Saccharomyces cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta Constructed by the CRISPR-Cas9 Approach with the Decrease of By-Product Formation

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