| 1. |
- Di Bartolomeo, Francesca, 1986, et al.
(författare)
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Absolute yeast mitochondrial proteome quantification reveals trade-off between biosynthesis and energy generation during diauxic shift
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:13, s. 7524-7535
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.
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| 2. |
- Cámara, Elena, 1985, et al.
(författare)
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Saccharomyces cerevisiae strains performing similarly during fermentation of lignocellulosic hydrolysates show pronounced differences in transcriptional stress responses
- 2024
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 90:5
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Tidskriftsartikel (refereegranskat)abstract
- Improving our understanding of the transcriptional changes of Saccharomyces cerevisiae during fermentation of lignocellulosic hydrolysates is crucial for the creation of more efficient strains to be used in biorefineries. We performed RNA sequencing of a CEN.PK laboratory strain, two industrial strains (KE6-12 and Ethanol Red), and two wild-type isolates of the LBCM collection when cultivated anaerobically in wheat straw hydrolysate. Many of the differently expressed genes identified among the strains have previously been reported to be important for tolerance to lignocellulosic hydrolysates or inhibitors therein. Our study demonstrates that stress responses typically identified during aerobic conditions such as glutathione metabolism, osmotolerance, and detoxification processes also are important for anaerobic processes. Overall, the transcriptomic responses were largely strain dependent, and we focused our study on similarities and differences in the transcriptomes of the LBCM strains. The expression of sugar transporter-encoding genes was higher in LBCM31 compared with LBCM109 that showed high expression of genes involved in iron metabolism and genes promoting the accumulation of sphingolipids, phospholipids, and ergosterol. These results highlight different evolutionary adaptations enabling S. cerevisiae to strive in lignocellulosic hydrolysates and suggest novel gene targets for improving fermentation performance and robustness.
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| 3. |
- Campbell, Kate, 1987, et al.
(författare)
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Building blocks are synthesized on demand during the yeast cell cycle
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:14, s. 7575-7583
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Tidskriftsartikel (refereegranskat)abstract
- For cells to replicate, a sufficient supply of biosynthetic precursors is needed, necessitating the concerted action of metabolism and protein synthesis during progressive phases of cell division. A global understanding of which biosynthetic processes are involved and how they are temporally regulated during replication is, however, currently lacking. Here, quantitative multiomics analysis is used to generate a holistic view of the eukaryal cell cycle, using the budding yeast Saccharomyces cerevisiae. Protein synthesis and central carbon pathways such as glycolysis and amino acid metabolism are shown to synchronize their respective abundance profiles with division, with pathway-specific changes in metabolite abundance also being reflected by a relative increase in mitochondrial volume, as shown by quantitative fluorescence microscopy. These results show biosynthetic precursor production to be temporally regulated to meet phase-specific demands of eukaryal cell division.
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| 4. |
- Mormino, Maurizio, 1988, et al.
(författare)
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Development of an Haa1-based biosensor for acetic acid sensing in Saccharomyces cerevisiae
- 2021
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 21:6
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Tidskriftsartikel (refereegranskat)abstract
- Acetic acid is one of the main inhibitors of lignocellulosic hydrolysates and acetic acid tolerance is crucial for the development of robust cell factories for conversion of biomass. As a precursor of acetyl-coenzyme A, it also plays an important role in central carbon metabolism. Thus, monitoring acetic acid levels is a crucial aspect when cultivating yeast. Transcription factor-based biosensors represent useful tools to follow metabolite concentrations. Here, we present the development of an acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1 that upon binding to acetic acid relocates to the nucleus. In the biosensor, a synthetic transcription factor consisting of Haa1 and BM3R1 from Bacillus megaterium was used to control expression of a reporter gene under a promoter containing BM3R1 binding sites. The biosensor did not drive expression under a promoter containing Haa1 binding sites and responded to acetic acid over a linear range spanning from 10 to 60 mM. To validate its applicability, the biosensor was integrated into acetic acid-producing strains. A direct correlation between biosensor output and acetic acid production was detected. The developed biosensor enables high-throughput screening of strains producing acetic acid and could also be used to investigate acetic acid-tolerant strain libraries.
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| 5. |
- Mormino, Maurizio, 1988
(författare)
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Exploring Saccharomyces cerevisiae’s responses to acetic acid and other inhibitors found in lignocellulosic hydrolysates
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The limited tolerance of the budding yeast Saccharomyces cerevisiae to lignocellulosic hydrolysate inhibitors is a key challenge to its use in biorefinery cell factories. Considerable resources have been invested in the isolation of yeast strains with better tolerance towards the inhibitors released during lignocellulose hydrolysis, such as acetic acid. The goal of this thesis was twofold: characterize the transcriptional response of S. cerevisiae to wheat straw hydrolysate and explore the role of essential S. cerevisiae genes in acetic and formic acid tolerance, using a new biosensor and competitive growth assays. The transcriptomes of one laboratory strain, two industrial strains, and two wild-type isolates grown in wheat straw hydrolysate were profiled. Despite similar growth, the isolates showed different expression of genes encoding proteins involved in oxidative stress response, lipid accumulation, and transport, suggesting different genetic strategies for tolerance. The new acetic acid biosensor was based on two transcription factors, Haa1 from S. cerevisiae and BM3R1 from Bacillus megaterium. Biosensor and competitive growth were used in parallel to screen a S. cerevisiae CRISPR interference strain library. While fluorescence-activated cell sorting led to the isolation of cells with higher acetic acid retention and sensitivity, competitive growth assays allowed the identification of cells with higher acid tolerance. The results confirmed the role in acid stress response of genes involved in glycogen accumulation, chromatin modification, and mitochondrial or proteasomal functions. Two novel targets for improving tolerance were also identified: PAP1 and HIP1. Altogether, this thesis provides mechanistic insight into the stress response to lignocellulosic hydrolysates or weak acid inhibitors that limit yeast-mediated conversion of lignocellulosic biomass into biochemicals. Additionally, it offers new tools for the identification of strains with altered acetic acid tolerance.
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| 6. |
- Mormino, Maurizio, 1988, et al.
(författare)
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Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library
- 2022
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 21:1, s. 214-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Acetic acid tolerance is crucial for the development of robust cell factories for conversion of lignocellulosic hydrolysates that typically contain high levels of acetic acid. Screening mutants for growth in medium with acetic acid is an attractive way to identify sensitive variants and can provide novel insights into the complex mechanisms regulating the acetic acid stress response. RESULTS: An acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1, was used to screen a CRISPRi yeast strain library where dCas9-Mxi was set to individually repress each essential or respiratory growth essential gene. Fluorescence-activated cell sorting led to the enrichment of a population of cells with higher acetic acid retention. These cells with higher biosensor signal were demonstrated to be more sensitive to acetic acid. Biosensor-based screening of the CRISPRi library strains enabled identification of strains with increased acetic acid sensitivity: strains with gRNAs targeting TIF34, MSN5, PAP1, COX10 or TRA1. CONCLUSIONS: This study demonstrated that biosensors are valuable tools for screening and monitoring acetic acid tolerance in yeast. Fine-tuning the expression of essential genes can lead to altered acetic acid tolerance.
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