Rare sugar D-allulose as a substitute sweetener is produced through the isomerization of D-fructose by D-tagatose 3-epimerases (DTEases) or D-allulose 3-epimerases (DAEases). D-Allulose is a kind of low energy monosaccharide sugar naturally existing in some fruits in very small quantities. D-Allulose not only possesses high value as a food ingredient and dietary supplement, but also exhibits a variety of physiological functions serving as improving insulin resistance, antioxidant enhancement, and hypoglycemic controls, and so forth. Thus, D-allulose has an important development value as an alternative to high-energy sugars. This review provided a systematic analysis of D-allulose characters, application, enzymatic characteristics and molecular modification, engineered strain construction, and processing technologies. The existing problems and its proposed solutions for D-allulose production are also discussed. More importantly, a green and recycling process technology for D-allulose production is proposed for low waste formation, low energy consumption, and high sugar yield.

The development of lignocellulosic bioethanol plays an important role in the substitution of petrochemical energy and high-value utilization of agricultural wastes. The safe and stable expression of cellulase gene sestc was achieved by applying the clustered regularly interspaced short palindromic repeats-Cas9 approach to the integration of sestc expression cassette containing Agaricus biporus glyceraldehyde-3-phosphate-dehydrogenase gene (gpd) promoter in the Saccharomyces cerevisiae chromosome. The target insertion site was found to be located in the S. cerevisiae hexokinase 2 by designing a gRNA expression vector. The recombinant SESTC protein exhibited a size of approximately 44 kDa in the engineered S. cerevisiae. By using orange peel as the fermentation substrate, the filter paper, endo-1,4-β-glucanase, exo-1,4-β-glucanase activities of the transformants were 1.06, 337.42, and 1.36 U/mL, which were 35.3-fold, 23.03-fold, and 17-fold higher than those from wild-type S. cerevisiae, respectively. After 6 h treatment, approximately 20 g/L glucose was obtained. Under anaerobic conditions the highest ethanol concentration reached 7.53 g/L after 48 h fermentation and was 37.7-fold higher than that of wild-type S. cerevisiae (0.2 g/L). The engineered strains may provide a valuable material for the development of lignocellulosic ethanol.

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.

Background: The thermotolerant yeast is beneficial in terms of efficiency improvement of processes and reduction of costs, while Saccharomyces cerevisiae does not efficiently grow and ferment at high-temperature conditions. The sterol composition alteration from ergosterol to fecosterol in the cell membrane of S. cerevisiae affects the thermotolerant capability. Results: In this study, S. cerevisiae ERG5, ERG4, and ERG3 were knocked out using the CRISPR–Cas9 approach to impact the gene expression involved in ergosterol synthesis. The highest thermotolerant strain was S. cerevisiae ERG5ΔERG4ΔERG3Δ, which produced 22.1 g/L ethanol at 37 °C using the initial glucose concentration of 50 g/L with an increase by 9.4% compared with the wild type (20.2 g/L). The ethanol concentration of 9.4 g/L was produced at 42 ℃, which was 2.85-fold of the wild-type strain (3.3 g/L). The molecular mechanism of engineered S. cerevisiae at the RNA level was analyzed using the transcriptomics method. The simultaneous deletion of S. cerevisiae ERG5, ERG4, and ERG3 caused 278 up-regulated genes and 1892 down-regulated genes in comparison with the wild-type strain. KEGG pathway analysis indicated that the up-regulated genes relevant to ergosterol metabolism were ERG1, ERG11, and ERG5, while the down-regulated genes were ERG9 and ERG26. S. cerevisiae ERG5ΔERG4ΔERG3Δ produced 41.6 g/L of ethanol at 37 °C with 107.7 g/L of corn liquefied glucose as carbon source. Conclusion: Simultaneous deletion of ERG5, ERG4, and ERG3 resulted in the thermotolerance improvement of S. cerevisiae ERG5ΔERG4ΔERG3Δ with cell viability improvement by 1.19-fold at 42 °C via modification of steroid metabolic pathway. S. cerevisiae ERG5ΔERG4ΔERG3Δ could effectively produce ethanol at 37 °C using corn liquefied glucose as carbon source. Therefore, S. cerevisiae ERG5ΔERG4ΔERG3Δ had potential in ethanol production at a large scale under supra-optimal temperature.