Dataset Information

ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production.

ABSTRACT:

Background

Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation. It also appears to be a suitable approach to improve production of ethanol by Saccharomyces cerevisiae. In the present study, we constructed different S. cerevisiae strains with heterologous expression of genes of the ATP-hydrolyzing F₁-part of the ATPase enzyme to induce enforced ATP wasting and quantify the resulting effect on biomass and ethanol formation.

Results

In contrast to genomic integration, we found that episomal expression of the ??? subunits of the F₁-ATPase genes of Escherichia coli in S. cerevisiae resulted in significantly increased ATPase activity, while neither genomic integration nor episomal expression of the ? subunit from Trichoderma reesei could enhance ATPase activity. When grown in minimal medium under anaerobic growth-coupled conditions, the strains expressing E. coli's F₁-ATPase genes showed significantly improved ethanol yield (increase of 10% compared to the control strain). However, elevated product formation reduces biomass formation and, therefore, volumetric productivity. We demonstrate that this negative effect can be overcome under growth-decoupled (nitrogen-starved) operation with high and constant biomass concentration. Under these conditions, which mimic the second (production) phase of a two-stage fermentation process, the ATPase-expressing strains showed significant improvement in volumetric productivity (up to 111%) compared to the control strain.

Conclusions

Our study shows that expression of genes of the F₁-portion of E. coli's ATPase induces ATPase activity in S. cerevisiae and can be a promising way to improve ethanol production. This ATP-wasting strategy can be easily applied to other metabolites of interest, whose formation is coupled to ATP generation.

SUBMITTER: Zahoor A

PROVIDER: S-EPMC7654063 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Publications

ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production.

Zahoor Ahmed A Messerschmidt Katrin K Boecker Simon S Klamt Steffen S

Biotechnology for biofuels 20201109 1

<h4>Background</h4>Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation. It also appears to be a suitable approach to improve production of ethanol by Saccharomyces cerevisiae. In the present study, we constructed different S. cerevisiae strains with heterologous expression of genes of the ATP-hydrolyzing F<sub>1</sub>-part of the ATPase enzyme to induce enforced ATP wasting an ...[more]

PMID: 33292464

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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.

Project description:BackgroundIn the yeast Saccharomyces cerevisiae, which is widely applied for industrial bioethanol production, uptake of hexoses is mediated by transporters with a facilitated diffusion mechanism. In anaerobic cultures, a higher ethanol yield can be achieved when transport of hexoses is proton-coupled, because of the lower net ATP yield of sugar dissimilation. In this study, the facilitated diffusion transport system for hexose sugars of S. cerevisiae was replaced by hexose-proton symport.ResultsIntroduction of heterologous glucose- or fructose-proton symporters in an hxt0 yeast background strain (derived from CEN.PK2-1C) restored growth on the corresponding sugar under aerobic conditions. After applying an evolutionary engineering strategy to enable anaerobic growth, the hexose-proton symporter-expressing strains were grown in anaerobic, hexose-limited chemostats on synthetic defined medium, which showed that the biomass yield of the resulting strains was decreased by 44.0-47.6%, whereas the ethanol yield had increased by up to 17.2% (from 1.51 to 1.77 mol mol hexose-1) compared to an isogenic strain expressing the hexose uniporter HXT5. To apply this strategy to increase the ethanol yield on sucrose, we constructed a platform strain in which all genes encoding hexose transporters, disaccharide transporters and disaccharide hydrolases were deleted, after which a combination of a glucose-proton symporter, fructose-proton symporter and extracellular invertase (SUC2) were introduced. After evolution, the resulting strain exhibited a 16.6% increased anaerobic ethanol yield (from 1.51 to 1.76 mol mol hexose equivalent-1) and 46.6% decreased biomass yield on sucrose.ConclusionsThis study provides a proof-of-concept for the replacement of the endogenous hexose transporters of S. cerevisiae by hexose-proton symport, and the concomitant decrease in ATP yield, to greatly improve the anaerobic yield of ethanol on sugar. Moreover, the sugar-negative platform strain constructed in this study acts as a valuable starting point for future studies on sugar transport or development of cell factories requiring specific sugar transport mechanisms.

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Dataset Information

ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production.

Background

Results

Conclusions

Publications

ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production.

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