Enhanced ethanol production via electrostatically accelerated fermentation of glucose using Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Mathew AS
PROVIDER: S-EPMC4626793 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:BackgroundBiofilm-immobilized continuous fermentation has the potential to enhance cellular environmental tolerance, maintain cell activity and improve production efficiency.ResultsIn this study, different biofilm-forming genes (FLO5, FLO8 and FLO10) were integrated into the genome of S. cerevisiae for overexpression, while FLO5 and FLO10 gave the best results. The biofilm formation of the engineered strains 1308-FLO5 and 1308-FLO10 was improved by 31.3% and 58.7% compared to that of the WT strain, respectively. The counts of cells adhering onto the biofilm carrier were increased. Compared to free-cell fermentation, the average ethanol production of 1308, 1308-FLO5 and 1308-FLO10 was increased by 17.4%, 20.8% and 19.1% in the biofilm-immobilized continuous fermentation, respectively. Due to good adhering ability, the fermentation broth turbidity of 1308-FLO5 and 1308-FLO10 was decreased by 22.3% and 59.1% in the biofilm-immobilized fermentation, respectively. Subsequently, for biofilm-immobilized fermentation coupled with membrane separation, the engineered strain significantly reduced the pollution of cells onto the membrane and the membrane separation flux was increased by 36.3%.ConclusionsIn conclusion, enhanced biofilm-forming capability of S. cerevisiae could offer multiple benefits in ethanol fermentation.
| S-EPMC10391976 | biostudies-literature
Project description:During fermentation, yeast cells encounter a number of stresses, including hyperosmolarity, high ethanol concentration, and high temperature. Previous deletome analysis in the yeast Saccharomyces cerevisiae has revealed that SOD1 gene encoding cytosolic Cu/Zn-superoxide dismutase (SOD), a major antioxidant enzyme, was required for tolerances to not only oxidative stress but also other stresses present during fermentation such as osmotic, ethanol, and heat stresses. It is therefore possible that these fermentation-associated stresses may also induce endogenous oxidative stress. In this study, we show that osmotic, ethanol, and heat stresses promoted generation of intracellular reactive oxygen species (ROS) such as superoxide anion in the cytosol through a mitochondria-independent mechanism. Consistent with this finding, cytosolic Cu/Zn-SOD, but not mitochondrial Mn-SOD, was required for protection against oxidative stress induced by these fermentation-associated stresses. Furthermore, supplementation of ROS scavengers such as N-acetyl-L-cysteine (NAC) alleviated oxidative stress induced during very high gravity (VHG) fermentation and enhanced fermentation performance at both normal and high temperatures. In addition, NAC also plays an important role in maintaining the Cu/Zn-SOD activity during VHG fermentation. These findings suggest the potential role of ROS scavengers for application in industrial-scale VHG ethanol fermentation.
| S-EPMC6117276 | biostudies-literature
Project description:BACKGROUND:Classical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes. RESULTS:Error-prone PCR was employed to engineer the subunit Rpb7 of RNAP II to improve yeast ethanol tolerance and production. Based on previous studies and the presumption that improved ethanol resistance would lead to enhanced ethanol production, we first isolated variant M1 with much improved resistance towards 8 and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response, etc. CONCLUSIONS:This is the first study to demonstrate the possibility of engineering eukaryotic RNAP to alter global transcription profile and improve strain phenotypes. Targeting subunit Rpb7 of RNAP II was able to bring differential expression in hundreds of genes in S. cerevisiae, which finally led to improvement in yeast ethanol tolerance and production.
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Project description:Saccharomyces cerevisiae is known for its outstanding ability to produce ethanol in industry. Underlying the dynamics of gene expression in S. cerevisiae in response to fermentation could provide informative results, required for the establishment of any ethanol production improvement program. Thus, representing a new approach, this study was conducted to identify the discriminative genes between improved and repressed ethanol production as well as clarifying the molecular responses to this process through mining the transcriptomic data. The significant differential expression probe sets were extracted from available microarray datasets related to yeast fermentation performance. To identify the most effective probe sets contributing to discriminate ethanol content, 11 machine learning algorithms from RapidMiner were employed. Further analysis including pathway enrichment and regulatory analysis were performed on discriminative probe sets. Besides, the decision tree models were constructed, the performance of each model was evaluated and the roots were identified. Based on the results, 171 probe sets were identified by at least 5 attribute weighting algorithms (AWAs) and 17 roots were recognized with 100% performance Some of the top ranked presets were found to be involved in carbohydrate metabolism, oxidative phosphorylation, and ethanol fermentation. Principal component analysis (PCA) and heatmap clustering validated the top-ranked selective probe sets. In addition, the top-ranked genes were validated based on GSE78759 and GSE5185 dataset. From all discriminative probe sets, OLI1 and CYC3 were identified as the roots with the best performance, demonstrated by the most weighting algorithms and linked to top two significant enriched pathways including porphyrin biosynthesis and oxidative phosphorylation. ADH5 and PDA1 were also recognized as differential top-ranked genes that contribute to ethanol production. According to the regulatory clustering analysis, Tup1 has a significant effect on the top-ranked target genes CYC3 and ADH5 genes. This study provides a basic understanding of the S. cerevisiae cell molecular mechanism and responses to two different medium conditions (Mg2+ and Cu2+) during the fermentation process.
| S-EPMC9321456 | biostudies-literature
Project description:Co-utilization of xylose and glucose from lignocellulosic biomass is an economically feasible bioprocess for chemical production. Many strategies have been implemented for efficiently assimilating xylose which is one of the predominant sugars of lignocellulosic biomass. However, there were few reports about engineering Saccharomyces cerevisiae for carotenoid production from xylose-glucose mixtures. Herein, we developed a platform for facilitating carotenoid production in S. cerevisiae by fermentation of xylose-glucose mixtures. Firstly, a xylose assimilation pathway with mutant xylose reductase (XYL1m), xylitol dehydrogenase (XYL2), and xylulokinase (XK) was constructed for utilizing xylose. Then, introduction of phosphoketolase (PK) pathway, deletion of Pho13 and engineering yeast hexose transporter Gal2 were conducted to improve carotenoid yields. The final strain SC105 produced a 1.6-fold higher production from mixed sugars than that from glucose in flask culture. In fed-batch fermentation with continuous feeding of mixed sugars, carotenoid production represented a 2.6-fold higher. To the best of our knowledge, this is the first report that S. cerevisiae was engineered to utilize xylose-glucose mixtures for carotenoid production with a considerable high yield. The present study exhibits a promising advantage of xylose-glucose mixtures assimilating strain as an industrial carotenoid producer from lignocellulosic biomass.
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Project description:Redox homeostasis is essential to the maintenance of cell metabolism. Changes in the redox state cause global metabolic and transcriptional changes. Our previous study indicated that the overexpression of NADH oxidase in Saccharomyces cerevisiae led to increased glucose consumption and ethanol production. Gene expression related to thiamine synthesis and osmotolerance as well as HAP4 expression was increased in response to redox change caused by the overexpression of NADH oxidase. To identify detailed relationships among cofactor levels, thiamine synthesis, expression of HAP4, and osmotolerance, and to determine whether these changes are interdependent, THI4 and HAP4 were overexpressed in S. cerevisiae BY4741. The glucose consumption rate of THI4-overexpressing strain (thi4-OE) was the highest, followed by HAP4-overexpressing strain (hap4-OE) > NADH oxidase-overexpressing strain (nox-OE) > control strain (con), while strain hap4-OE showed the highest concentration of ethanol after 26 h of fermentation. Reduced glycerol production and increased osmotolerance were observed in thi4-OE and hap4-OE, as well as in nox-OE. HAP4 globally regulated thiamine synthesis, biomass synthesis, respiration, and osmotolerance of cells, which conferred the recombinant strain hap4-OE with faster glucose metabolism and enhanced stress resistance. Moreover, overexpression of HAP4 might extend the life span of cells under caloric restriction by lowering the NADH level. Although overexpression of THI4 and HAP4 induced various similar changes at both the metabolic and the transcriptional level, the regulatory effect of THI4 was more limited than that of HAP4, and was restricted to the growth phase of cells. Our findings are expected to benefit the bio-ethanol industry.
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Project description:Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.
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Project description:Replacement of a novel candidate ethanol fermentation-associated regulatory gene, PHO4, from a fast-growing strain MC15, as determined through comparative genomics analysis among three yeast strains with significant differences in ethanol yield, is hypothesised to shorten the fermentation time and enhance ethanol production from sugarcane molasses. This study sought to test this hypothesis through a novel strategy involving the transfer of the PHO4 gene from a low ethanol-producing, yet fast-growing strain MC15 to a high ethanol-producing industrial strain MF01 through homologous recombination. The results indicated that PHO4 in the industrially engineered strain MF01-PHO4 displayed genomic stability with a mean maximum ethanol yield that rose to 114.71 g L-1, accounting for a 5.30% increase in ethanol yield and 12.5% decrease in fermentation time in comparison with that in the original strain MF01, which was the current highest ethanol-producing strain in SCM fermentation in the reported literature. These results serve to advance our current understanding of the association between improving ethanol yield and replacement of PHO4, while providing a feasible strategy for industrially engineered yeast strains to improve ethanol production efficiently.
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Project description:BackgroundCost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck.ResultsMulti-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively at 20 and 30 °C. Maximum taxadiene titres of 129 ± 15 mg/L and 127 mg/L were achieved through shake flask and bioreactor cultivations, respectively, of the optimal strain at a reduced temperature of 20 °C.ConclusionsThe results of this study highlight the benefit of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5-fold compared to the highest literature titre in S. cerevisiae cell factories.
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Project description:Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO2 production and final ethanol concentration generated by the atg32? laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of the atg32? mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32? mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts.
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