Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor

ABSTRACT In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde + NAD+ + coenzyme A ↔ acetyl coenzyme A + NADH + H+), was expressed in the gpd1Δ gpd2Δ strain, anaerobic growth was restored by supplementation with 2.0 g liter−1 acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).

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Effects of Furfural on the Respiratory Metabolism of Saccharomyces cerevisiae in Glucose-Limited Chemostats

Applied and Environmental Microbiology ◽

10.1128/aem.69.7.4076-4086.2003 ◽

2003 ◽

Vol 69 (7) ◽

pp. 4076-4086 ◽

Cited By ~ 98

Author(s):

Ilona Sárvári Horváth ◽

Carl Johan Franzén ◽

Mohammad J. Taherzadeh ◽

Claes Niklasson ◽

Gunnar Lidén

Keyword(s):

Saccharomyces Cerevisiae ◽

Furfuryl Alcohol ◽

Inhibitory Effect ◽

Anaerobic Growth ◽

Yeast Cells ◽

Flux Analysis ◽

Complete Replacement ◽

Furoic Acid ◽

Conversion Rates ◽

Glycerol Formation

ABSTRACT Effects of furfural on the aerobic metabolism of the yeast Saccharomyces cerevisiae were studied by performing chemostat experiments, and the kinetics of furfural conversion was analyzed by performing dynamic experiments. Furfural, an important inhibitor present in lignocellulosic hydrolysates, was shown to have an inhibitory effect on yeast cells growing respiratively which was much greater than the inhibitory effect previously observed for anaerobically growing yeast cells. The residual furfural concentration in the bioreactor was close to zero at all steady states obtained, and it was found that furfural was exclusively converted to furoic acid during respiratory growth. A metabolic flux analysis showed that furfural affected fluxes involved in energy metabolism. There was a 50% increase in the specific respiratory activity at the highest steady-state furfural conversion rate. Higher furfural conversion rates, obtained during pulse additions of furfural, resulted in respirofermentative metabolism, a decrease in the biomass yield, and formation of furfuryl alcohol in addition to furoic acid. Under anaerobic conditions, reduction of furfural partially replaced glycerol formation as a way to regenerate NAD+. At concentrations above the inlet concentration of furfural, which resulted in complete replacement of glycerol formation by furfuryl alcohol production, washout occurred. Similarly, when the maximum rate of oxidative conversion of furfural to furoic acid was exceeded aerobically, washout occurred. Thus, during both aerobic growth and anaerobic growth, the ability to tolerate furfural appears to be directly coupled to the ability to convert furfural to less inhibitory compounds.

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Saccharomyces cerevisiae Fermentation of 28 Barley and 12 Oat Cultivars

Fermentation ◽

10.3390/fermentation7020059 ◽

2021 ◽

Vol 7 (2) ◽

pp. 59

Author(s):

Timothy J. Tse ◽

Daniel J. Wiens ◽

Jianheng Shen ◽

Aaron D. Beattie ◽

Martin J. T. Reaney

Keyword(s):

Saccharomyces Cerevisiae ◽

Acetic Acid ◽

Lactic Acid ◽

Succinic Acid ◽

Alcoholic Fermentation ◽

Ethanol Yield ◽

Cereal Crops ◽

Fermentation Products ◽

Barley Cultivars ◽

Oat Cultivars

As barley and oat production have recently increased in Canada, it has become prudent to investigate these cereal crops as potential feedstocks for alcoholic fermentation. Ethanol and other coproduct yields can vary substantially among fermented feedstocks, which currently consist primarily of wheat and corn. In this study, the liquified mash of milled grains from 28 barley (hulled and hull-less) and 12 oat cultivars were fermented with Saccharomyces cerevisiae to determine concentrations of fermentation products (ethanol, isopropanol, acetic acid, lactic acid, succinic acid, α-glycerylphosphorylcholine (α-GPC), and glycerol). On average, the fermentation of barley produced significantly higher amounts of ethanol, isopropanol, acetic acid, succinic acid, α-GPC, and glycerol than that of oats. The best performing barley cultivars were able to produce up to 78.48 g/L (CDC Clear) ethanol and 1.81 g/L α-GPC (CDC Cowboy). Furthermore, the presence of milled hulls did not impact ethanol yield amongst barley cultivars. Due to its superior ethanol yield compared to oats, barley is a suitable feedstock for ethanol production. In addition, the accumulation of α-GPC could add considerable value to the fermentation of these cereal crops.

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Translation machinery reprogramming in programmed cell death in Saccharomyces cerevisiae

Cell Death Discovery ◽

10.1038/s41420-020-00392-x ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Francesco Monticolo ◽

Emanuela Palomba ◽

Maria Luisa Chiusano

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Acetic Acid ◽

Programmed Cell Death ◽

Differential Expression ◽

Ribosomal Proteins ◽

Relative Proportion ◽

Duplication Event ◽

Genome Duplication Event ◽

Cell Death Pathway

AbstractProgrammed cell death involves complex molecular pathways in both eukaryotes and prokaryotes. In Escherichia coli, the toxin–antitoxin system (TA-system) has been described as a programmed cell death pathway in which mRNA and ribosome organizations are modified, favoring the production of specific death-related proteins, but also of a minor portion of survival proteins, determining the destiny of the cell population. In the eukaryote Saccharomyces cerevisiae, the ribosome was shown to change its stoichiometry in terms of ribosomal protein content during stress response, affecting the relative proportion between ohnologs, i.e., the couple of paralogs derived by a whole genome duplication event. Here, we confirm the differential expression of ribosomal proteins in yeast also during programmed cell death induced by acetic acid, and we highlight that also in this case pairs of ohnologs are involved. We also show that there are different trends in cytosolic and mitochondrial ribosomal proteins gene expression during the process. Moreover, we show that the exposure to acetic acid induces the differential expression of further genes coding for products related to translation processes and to rRNA post-transcriptional maturation, involving mRNA decapping, affecting translation accuracy, and snoRNA synthesis. Our results suggest that the reprogramming of the overall translation apparatus, including the cytosolic ribosome reorganization, are relevant events in yeast programmed cell death induced by acetic acid.

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Regulatory Mechanisms Controlling Expression of the DAN/TIR Mannoprotein Genes During Anaerobic Remodeling of the Cell Wall in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.3.1169 ◽

2001 ◽

Vol 157 (3) ◽

pp. 1169-1177

Author(s):

Natalia E Abramova ◽

Brian D Cohen ◽

Odeniel Sertil ◽

Rachna Kapoor ◽

Kelvin J A Davies ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Consensus Sequence ◽

Ectopic Expression ◽

Constitutive Expression ◽

Anaerobic Growth ◽

Zinc Cluster ◽

Sterol Transport ◽

Genes Encoding ◽

Altered Regulation ◽

Repression Domain

Abstract The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Δ allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.

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Eukaryotic translation factor eIF5A contributes to acetic acid tolerance in Saccharomyces cerevisiae via transcriptional factor Ume6p

Biotechnology for Biofuels ◽

10.1186/s13068-021-01885-2 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yanfei Cheng ◽

Hui Zhu ◽

Zhengda Du ◽

Xuena Guo ◽

Chenyao Zhou ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Acetic Acid ◽

Adaptive Response ◽

Acid Tolerance ◽

Peptide Bond ◽

Transcriptional Factor ◽

Yeast Cells ◽

Acetic Acid Tolerance ◽

Translation Factor ◽

Industrial Strains

Abstract Background Saccharomyces cerevisiae is well-known as an ideal model system for basic research and important industrial microorganism for biotechnological applications. Acetic acid is an important growth inhibitor that has deleterious effects on both the growth and fermentation performance of yeast cells. Comprehensive understanding of the mechanisms underlying S. cerevisiae adaptive response to acetic acid is always a focus and indispensable for development of robust industrial strains. eIF5A is a specific translation factor that is especially required for the formation of peptide bond between certain residues including proline regarded as poor substrates for slow peptide bond formation. Decrease of eIF5A activity resulted in temperature-sensitive phenotype of yeast, while up-regulation of eIF5A protected transgenic Arabidopsis against high temperature, oxidative or osmotic stress. However, the exact roles and functional mechanisms of eIF5A in stress response are as yet largely unknown. Results In this research, we compared cell growth between the eIF5A overexpressing and the control S. cerevisiae strains under various stressed conditions. Improvement of acetic acid tolerance by enhanced eIF5A activity was observed all in spot assay, growth profiles and survival assay. eIF5A prompts the synthesis of Ume6p, a pleiotropic transcriptional factor containing polyproline motifs, mainly in a translational related way. As a consequence, BEM4, BUD21 and IME4, the direct targets of Ume6p, were up-regulated in eIF5A overexpressing strain, especially under acetic acid stress. Overexpression of UME6 results in similar profiles of cell growth and target genes transcription to eIF5A overexpression, confirming the role of Ume6p and its association between eIF5A and acetic acid tolerance. Conclusion Translation factor eIF5A protects yeast cells against acetic acid challenge by the eIF5A-Ume6p-Bud21p/Ime4p/Bem4p axles, which provides new insights into the molecular mechanisms underlying the adaptive response and tolerance to acetic acid in S. cerevisiae and novel targets for construction of robust industrial strains.

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A genome-scale metabolic model of Saccharomyces cerevisiae that integrates expression constraints and reaction thermodynamics

Nature Communications ◽

10.1038/s41467-021-25158-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Omid Oftadeh ◽

Pierre Salvy ◽

Maria Masid ◽

Maxime Curvat ◽

Ljubisa Miskovic ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Expression System ◽

Biomass Composition ◽

Industrial Biotechnology ◽

Overflow Metabolism ◽

Cellular Expression ◽

A Genome ◽

Wide Range ◽

Eukaryotic Organisms ◽

Genome Scale

AbstractEukaryotic organisms play an important role in industrial biotechnology, from the production of fuels and commodity chemicals to therapeutic proteins. To optimize these industrial systems, a mathematical approach can be used to integrate the description of multiple biological networks into a single model for cell analysis and engineering. One of the most accurate models of biological systems include Expression and Thermodynamics FLux (ETFL), which efficiently integrates RNA and protein synthesis with traditional genome-scale metabolic models. However, ETFL is so far only applicable for E. coli. To adapt this model for Saccharomyces cerevisiae, we developed yETFL, in which we augmented the original formulation with additional considerations for biomass composition, the compartmentalized cellular expression system, and the energetic costs of biological processes. We demonstrated the ability of yETFL to predict maximum growth rate, essential genes, and the phenotype of overflow metabolism. We envision that the presented formulation can be extended to a wide range of eukaryotic organisms to the benefit of academic and industrial research.

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Sphingolipid biosynthesis upregulation by TOR complex 2–Ypk1 signaling during yeast adaptive response to acetic acid stress

Biochemical Journal ◽

10.1042/bcj20160565 ◽

2016 ◽

Vol 473 (23) ◽

pp. 4311-4325 ◽

Cited By ~ 27

Author(s):

Joana F. Guerreiro ◽

Alexander Muir ◽

Subramaniam Ramachandran ◽

Jeremy Thorner ◽

Isabel Sá-Correia

Keyword(s):

Acetic Acid ◽

Protein Kinase ◽

Cellular Response ◽

Molecular Mechanisms ◽

Acid Stress ◽

Acid Tolerance ◽

Industrial Biotechnology ◽

Acetic Acid Tolerance ◽

Spoilage Yeasts ◽

Sphingolipid Biosynthesis

Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2–Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2–Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2–Ypk1–sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks.

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