| 1. |
- Trivellin, Cecilia, 1993, et al.
(författare)
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Robustness quantification of a mutant library screen revealed key genetic markers in yeast
- 2024
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Ingår i: Microbial Cell Factories. - 1475-2859. ; 23:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Microbial robustness is crucial for developing cell factories that maintain consistent performance in a challenging environment such as large-scale bioreactors. Although tools exist to assess and understand robustness at a phenotypic level, the underlying metabolic and genetic mechanisms are not well defined, which limits our ability to engineer more strains with robust functions. Results: This study encompassed four steps. (I) Fitness and robustness were analyzed from a published dataset of yeast mutants grown in multiple environments. (II) Genes and metabolic processes affecting robustness or fitness were identified, and 14 of these genes were deleted in Saccharomyces cerevisiae CEN.PK113-7D. (III) The mutants bearing gene deletions were cultivated in three perturbation spaces mimicking typical industrial processes. (IV) Fitness and robustness were determined for each mutant in each perturbation space. We report that robustness varied according to the perturbation space. We identified genes associated with increased robustness such as MET28, linked to sulfur metabolism; as well as genes associated with decreased robustness, including TIR3 and WWM1, both involved in stress response and apoptosis. Conclusion: The present study demonstrates how phenomics datasets can be analyzed to reveal the relationship between phenotypic response and associated genes. Specifically, robustness analysis makes it possible to study the influence of single genes and metabolic processes on stable microbial performance in different perturbation spaces. Ultimately, this information can be used to enhance robustness in targeted strains.
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| 2. |
- Ask, Magnus, 1983, et al.
(författare)
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The influence of HMF and furfural on redox-balance and energy-state of xylose-utilizing Saccharomyces cerevisiae
- 2013
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 6:22
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundPretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. The furan aldehydes hydroxymethylfurfural (HMF) and furfural have received increasing attention recently. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and gene expression were investigated in anaerobic chemostats where the inhibitors were added to the feed-medium.ResultsBy cultivating the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 in the presence of HMF and furfural, it was found that the intracellular concentrations of the redox co-factors and the catabolic and anabolic reduction charges were significantly lower in the presence of furan aldehydes than in cultivations without inhibitors. The catabolic reduction charge decreased from 0.13(+/-0.005) to 0.08(+/-0.002) and the anabolic reduction charge decreased from 0.46(+/-0.11) to 0.27(+/-0.02) when HMF and furfural were present. The intracellular ATP concentration was lower when inhibitors were added, but resulted only in a modest decrease in the energy charge from 0.87(+/-0.002) to 0.85(+/-0.004) compared to the control. Transcriptome profiling followed by MIPS functional enrichment analysis of up-regulated genes revealed that the functional group "Cell rescue, defense and virulence" was over-represented when inhibitors were present compared to control cultivations. Among these, the ATP-binding efflux pumps PDR5 and YOR1 were identified as important for inhibitor efflux and possibly a reason for the lower intracellular ATP concentration in stressed cells. It was also found that genes involved in pseudohyphal growth were among the most up-regulated when inhibitors were present in the feed-medium suggesting nitrogen starvation. Genes involved in amino acid metabolism, glyoxylate cycle, electron transport and amino acid transport were enriched in the down-regulated gene set in response to HMF and furfural. It was hypothesized that the HMF and furfural-induced NADPH drainage could influence ammonia assimilation and thereby give rise to the nitrogen starvation response in the form of pseudohyphal growth and down-regulation of amino acid synthesis.ConclusionsThe redox metabolism was severely affected by HMF and furfural while the effects on energy metabolism were less evident, suggesting that engineering of the redox system represents a possible strategy to develop more robust strains for bioethanol production.
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| 3. |
- Bettiga, Maurizio, 1978, et al.
(författare)
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Plasma membrane as a crucial player in acetic acid effect on yeast
- 2017
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Ingår i: IMYA12- 12th International Meeting on Yeast Apoptosis, Bari, Italy • 14-18 May 2017.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Weak organic acids such as formic, acetic or lactic acid are known inhibitors of microbial growth and fermentation. Acetic acid toxicity to yeast cells has been explained by different theories, involving specific signaling effects triggering an active cell death program, reduction of intracellular pH and acetate anion accumulation. Regardless of the fact whether the actual effect of acetate involves one of these mechanisms or a combination thereof, acetic acid inhibits yeast metabolism and affects yeast viability. This has a high impact on the feasibility of the new generation of fermentation processes, based on the naturally acetic acid-rich lignocellulosic substrates. It is therefore highly desirable to obtain a strain with increased capacity of coping with high acetic acid concentrations in the fermentation medium. Acetic acid is thought to be internalized by yeast cells in its undissociated form, by crossing the hydrophobic barrier of plasma membrane. Thus, in our work we focused on the investigation of membrane properties and how these influence the tolerance of yeast to acetic acid. First, we demonstrated with lipidomics analysis of membrane lipids that the yeast Zygosaccharomyces bailii, showing extraordinary tolerance to acetic acid, has a plasma membrane which is rich in sphingolipids. Next, we combined membrane molecular dynamics and in vivo measurements to confirm the specific role of sphingolipids in altering the permeability of plasma membrane to acetic acid. Finally, we investigated the effect of alcohols on the acetic acid permeation rate through the membrane. Our ultimate goal is to engineer the membrane composition of an industrial yeast strain towards reduced permeability, in order to obtain higher acetic acid tolerance.
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| 4. |
- Vu Nguyen Thanh,, et al.
(författare)
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Surveying of acid-tolerant thermophilic lignocellulolytic fungi in Vietnam reveals surprisingly high genetic diversity
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Thermophilic fungi can represent a rich source of industrially relevant enzymes. Here, 105 fungal strains capable of growing at 50 degrees C and pH 2.0 were isolated from compost and decaying plant matter. Maximum growth temperatures of the strains were in the range 50 degrees C to 60 degrees C. Sequencing of the internal transcribed spacer (ITS) regions indicated that 78 fungi belonged to 12 species of Ascomycota and 3 species of Zygomycota, while no fungus of Basidiomycota was detected. The remaining 27 strains could not be reliably assigned to any known species. Phylogenetically, they belonged to the genus Thielavia, but they represented 23 highly divergent genetic groups different from each other and from the closest known species by 12 to 152 nucleotides in the ITS region. Fungal secretomes of all 105 strains produced during growth on untreated rice straw were studied for lignocellulolytic activity at different pH and temperatures. The endoglucanase and xylanase activities differed substantially between the different species and strains, but in general, the enzymes produced by the novel Thielavia spp. strains exhibited both higher thermal stability and tolerance to acidic conditions. The study highlights the vast potential of an untapped diversity of thermophilic fungi in the tropics.
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| 5. |
- Arnling Bååth, Jenny, 1987, et al.
(författare)
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Biochemical and structural features of diverse bacterial glucuronoyl esterases facilitating recalcitrant biomass conversion
- 2018
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Lignocellulose is highly recalcitrant to enzymatic deconstruction, where the recalcitrance primarily results from chemical linkages between lignin and carbohydrates. Glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15) have been suggested to play key roles in reducing lignocellulose recalcitrance by cleaving covalent ester bonds found between lignin and glucuronoxylan. However, only a limited number of GEs have been biochemically characterized and structurally determined to date, limiting our understanding of these enzymes and their potential exploration. Results Ten CE15 enzymes from three bacterial species, sharing as little as 20% sequence identity, were characterized on a range of model substrates; two protein structures were solved, and insights into their regulation and biological roles were gained through gene expression analysis and enzymatic assays on complex biomass. Several enzymes with higher catalytic efficiencies on a wider range of model substrates than previously characterized fungal GEs were identified. Similarities and differences regarding substrate specificity between the investigated GEs were observed and putatively linked to their positioning in the CE15 phylogenetic tree. The bacterial GEs were able to utilize substrates lacking 4-OH methyl substitutions, known to be important for fungal enzymes. In addition, certain bacterial GEs were able to efficiently cleave esters of galacturonate, a functionality not previously described within the family. The two solved structures revealed similar overall folds to known structures, but also indicated active site regions allowing for more promiscuous substrate specificities. The gene expression analysis demonstrated that bacterial GE-encoding genes were differentially expressed as response to different carbon sources. Further, improved enzymatic saccharification of milled corn cob by a commercial lignocellulolytic enzyme cocktail when supplemented with GEs showcased their synergistic potential with other enzyme types on native biomass. Conclusions Bacterial GEs exhibit much larger diversity than fungal counterparts. In this study, we significantly expanded the existing knowledge on CE15 with the in-depth characterization of ten bacterial GEs broadly spanning the phylogenetic tree, and also presented two novel enzyme structures. Variations in transcriptional responses of CE15-encoding genes under different growth conditions suggest nonredundant functions for enzymes found in species with multiple CE15 genes and further illuminate the importance of GEs in native lignin–carbohydrate disassembly.
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| 6. |
- Brander, Søren, et al.
(författare)
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Biochemical evidence of both copper chelation and oxygenase activity at the histidine brace
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear copper site. This site is defined by an N-terminal histidine and a second internal histidine side chain in a configuration called the histidine brace. To understand better the determinants of reactivity, the biochemical and structural properties of a well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) is compared with that of CopC from Pseudomonas fluorescens (PfCopC) and with the LPMO-like protein Bim1 from Cryptococcus neoformans. PfCopC is not reduced by ascorbate but is a very strong Cu(II) chelator due to residues that interacts with the N-terminus. This first biochemical characterization of Bim1 shows that it is not redox active, but very sensitive to H2O2, which accelerates the release of Cu ions from the protein. TaAA9A oxidizes ascorbate at a rate similar to free copper but through a mechanism that produce fewer reactive oxygen species. These three biologically relevant examples emphasize the diversity in how the proteinaceous environment control reactivity of Cu with O2.
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| 7. |
- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(författare)
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Genomic and transcriptomic analysis of Candida intermedia reveals genes for utilization of biotechnologically important carbon sources
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A future biobased society relies on efficient industrial microorganisms that can convert all sugars from agricultural, forestry and industrial waste streams into fuels, chemicals and materials. To be able to tailor-make such potent cell factories, we need a far better understanding of the proteins responsible for the assimilation of biotechnologically important carbon sources including pentoses, disaccharides and oligomers. The yeast Candida intermedia, known for its superior growth on xylose owing to its efficient uptake and conversion systems, can also utilize a range of other important carbon sources such as cellobiose, galactose and lactose. The aim of this project was to identify the genomic determinants for the utilization of these mono- and disaccharides in our in-house isolated C. intermedia strain CBS 141442. Genome sequencing and transcriptional (RNA seq) data analysis during growth in defined medium supplemented with glucose, xylose, galactose, lactose or cellobiose, revealed numerous distinct clusters of coregulated genes. By scanning the CBS 141442 genome for genes encoding Major Facilitator Superfamily (MFS) sugar transporters, and the RNA-seq dataset for the corresponding transcriptional profiles, we identified several novel genes encoding putative xylose transporters and multiple Lac12-like transporters likely involved in the uptake of disaccharides in C. intermedia. We also found that the yeast possesses no less than three genes encoding aldose reductases with different transcriptional profiles, and heterologous expression of the genes in Saccharomyces cerevisiae showed that the aldose reductases have different substrate and co-factor specificities, suggesting diverse physiological roles. Taken together, the results of this study provide insights into the mechanisms underlying carbohydrate metabolism in C. intermedia, and reveals several genes with potential future applications in cell factory development.
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| 8. |
- Da Silva Faria Oliveira, Fábio Luis, 1985, et al.
(författare)
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Split-marker recombination for efficient targeted gene deletions in Candida intermedia
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Candida intermedia is a non-conventional yeast species with a natural ability to produce ethanol from xylose, making it an attractive non-GMO alternative for lignocellulosic biomass conversion in biorefineries and/or gene donor to Saccharomyces cerevisiae to improve its xylose fermentation capacity. We have de novo genome sequenced the C. intermedia strain CBS 141442, previously isolated in our lab, which allows us to study the yeast at a genomic and molecular level. The aim of this project was to develop a molecular toolbox for C. intermedia to enable also targeted genome editing and subsequent mutant phenotyping. C. intermedia is a haploid yeast belonging to the CTG clade of fungal species, and thus requires drug-resistant markers adapted for the alternative codon usage of these organisms. Transformation of linearized plasmid containing the CaNAT1 marker flanked by the TEF1 promoter and terminator from Ashbya gossypii [1] resulted in hundreds of Nourseothricin-resistant transformants. We then constructed an ADE2-deletion cassette, where the CaNAT1 marker was fused to the upstream and downstream sequences (1000bp) of CiADE2. Transformations resulted in less than 1% of ade2 mutants with the characteristic red pigmentation, which indicates that the non-homologous end joining pathway (NHEJ) is dominant over the homologous recombination (HR) pathway in this yeast. Using the cell cycle inhibitor hydroxyurea to arrest cells in the S-phase has been shown to improve the HR/NHEJ ratio in other yeasts [2], and increased the ADE2 deletion efficiency to 4% in C. intermedia. To further improve the targeted deletion rate, we applied the "split-marker” strategy previously developed for Saccharomyces cerevisiae [3]. Here, the selectable marker gene is truncated in two different fragments, and the gene is not functional until homologous recombination takes place between the two overlapping parts of the fragments. The truncated marker gene fragments were flanked by homologous sequences (1000 bp) upstream and downstream of the target gene using fusion PCR, thereby avoiding a tedious cloning step. This approach increased the targeted gene disruption of ADE2 to 56%. As proof of concept, the method was also used to delete KU70, the xylose reductase gene XYL1_2 as well as a large gene cluster in C. intermedia, with allele-specific HR efficiencies between 87 and 100%. The split-marker approach for targeted gene-disruptions will pave the way for high throughput genetic analysis in C. intermedia as well as in other yeasts where NHEJ is the predominant form of recombination.
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| 9. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Genomic and transcriptomic analysis of Candida intermedia reveals the genetic determinants for its xylose-converting capacity
- 2020
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background An economically viable production of biofuels and biochemicals from lignocellulose requires microorganisms that can readily convert both the cellulosic and hemicellulosic fractions into product. The yeast Candida intermedia displays a high capacity for uptake and conversion of several lignocellulosic sugars including the abundant pentose d-xylose, an underutilized carbon source since most industrially relevant microorganisms cannot naturally ferment it. Thus, C. intermedia constitutes an important source of knowledge and genetic information that could be transferred to industrial microorganisms such as Saccharomyces cerevisiae to improve their capacity to ferment lignocellulose-derived xylose. Results To understand the genetic determinants that underlie the metabolic properties of C. intermedia, we sequenced the genomes of both the in-house-isolated strain CBS 141442 and the reference strain PYCC 4715. De novo genome assembly and subsequent analysis revealed C. intermedia to be a haploid species belonging to the CTG clade of ascomycetous yeasts. The two strains have highly similar genome sizes and number of protein-encoding genes, but they differ on the chromosomal level due to numerous translocations of large and small genomic segments. The transcriptional profiles for CBS 141442 grown in medium with either high or low concentrations of glucose and xylose were determined through RNA-sequencing analysis, revealing distinct clusters of co-regulated genes in response to different specific growth rates, carbon sources and osmotic stress. Analysis of the genomic and transcriptomic data also identified multiple xylose reductases, one of which displayed dual NADH/NADPH co-factor specificity that likely plays an important role for co-factor recycling during xylose fermentation. Conclusions In the present study, we performed the first genomic and transcriptomic analysis of C. intermedia and identified several novel genes for conversion of xylose. Together the results provide insights into the mechanisms underlying saccharide utilization in C. intermedia and reveal potential target genes to aid in xylose fermentation in S. cerevisiae.
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| 10. |
- Granchi, Zoraide, et al.
(författare)
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Genome Sequence of the Thermophilic Biomass-Degrading Fungus Malbranchea cinnamomea FCH 10.5
- 2017
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Ingår i: Genome Announcements. - 2169-8287. ; 5:33
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Tidskriftsartikel (refereegranskat)abstract
- We report here the annotated draft genome sequence of the thermophilic biomass-degrading fungus Malbranchea cinnamomea strain FCH 10.5, isolated from compost at a waste treatment plant in Vietnam. The genome sequence contains 24.96 Mb with an overall GC content of 49.79% and comprises 9,437 protein-coding genes.
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