Background: Saccharomyces cerevisiae generally consumes glucose to produce ethanol accompanied by the main by-products of glycerol, acetic acid, and lactic acid. The minimization of the formation of by-products in S. cerevisiae was an effective way to improve the economic viability of the bioethanol industry. In this study, S. cerevisiae GPD2, FPS1, ADH2, and DLD3 genes were knocked out by the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach. The mechanism of gene deletion affecting ethanol metabolism was further elucidated based on metabolic flux and transcriptomics approaches. Results: The engineered S. cerevisiae with gene deletion of GPD2, FPS1, ADH2, and DLD3 was constructed by the CRISPR-Cas9 approach. The ethanol content of engineered S. cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta DLD3 Delta increased by 18.58% with the decrease of glycerol, acetic acid, and lactic acid contents by 22.32, 8.87, and 16.82%, respectively. The metabolic flux analysis indicated that the carbon flux r_ethanol in engineered strain increased from 60.969 to 63.379. The sequencing-based RNA-Seq transcriptomics represented 472 differential expression genes (DEGs) were identified in engineered S. cerevisiae, in which 195 and 277 genes were significantly up-regulated and down-regulated, respectively. The enriched pathways of up-regulated genes were mainly involved in the energy metabolism of carbohydrates, while the down-regulated genes were mainly enriched in acid metabolic pathways. Conclusions: The yield of ethanol in engineered S. cerevisiae increased with the decrease of the by-products including glycerol, acetic acid, and lactic acid. The deletion of genes GPD2, FPS1, ADH2, and DLD3 resulted in the redirection of carbon flux.

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.

Aflatoxins seriously threaten the health of humans and animals due to their potential carcinogenic properties. Enzymatic degradation approach is an effective and environmentally friendly alternative that involves changing the structure of aflatoxins. In this study, Trametes versicolor aflatoxin B₁-degrading enzyme gene (TV-AFB₁D) was integrated into the genome of Pichia pastoris GS115 by homologous recombination approach. The recombinant TV-AFB₁D was expressed in engineering P. pastoris with a size of approximately 77 kDa under the induction of methanol. The maximum activity of TV-AFB₁D reached 17.5 U/mL after the induction of 0.8% ethanol (v/v) for 84 h at 28 °C. The AFB₁ proportion of 75.9% was degraded using AFB₁ standard sample after catalysis for 12 h. In addition, the AFB₁ proportion was 48.5% using AFB₁-contaminated peanuts after the catalysis for 18 h at 34 °C. The recombinant TV-AFB₁D would have good practical application value in AFB₁ degradation in food crops. This study provides an alternative degrading enzyme for the degradation of AFB₁ in aflatoxin-contaminated grain and feed via enzymatic degradation approach.