SwePub
Sök i SwePub databas

  Utökad sökning

Träfflista för sökning "WFRF:(Siewers Verena 1976) "

Sökning: WFRF:(Siewers Verena 1976)

  • Resultat 1-50 av 122
Sortera/gruppera träfflistan
   
NumreringReferensOmslagsbildHitta
1.
  • Otto, Maximilian, 1991, et al. (författare)
  • Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries
  • 2021
  • Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:12, s. 3461-3474
  • Tidskriftsartikel (refereegranskat)abstract
    • Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community.
  •  
2.
  •  
3.
  • Baumann, Leonie, et al. (författare)
  • Transcriptomic response of Saccharomyces cerevisiae to octanoic acid production
  • 2021
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 21:2
  • Tidskriftsartikel (refereegranskat)abstract
    • The medium-chain fatty acid octanoic acid is an important platform compound widely used in industry. The microbial production from sugars in Saccharomyces cerevisiae is a promising alternative to current non-sustainable production methods, however, titers need to be further increased. To achieve this, it is essential to have in-depth knowledge about the cell physiology during octanoic acid production. To this end, we collected the first RNA-Seq data of an octanoic acid producer strain at three time points during fermentation. The strain produced higher levels of octanoic acid and increased levels of fatty acids of other chain lengths (C6-C18) but showed decreased growth compared to the reference. Furthermore, we show that the here analyzed transcriptomic response to internally produced octanoic acid is notably distinct from a wild type's response to externally supplied octanoic acid as reported in previous publications. By comparing the transcriptomic response of different sampling times, we identified several genes that we subsequently overexpressed and knocked out, respectively. Hereby we identified RPL40B, to date unknown to play a role in fatty acid biosynthesis or medium-chain fatty acid tolerance. Overexpression of RPL40B led to an increase in octanoic acid titers by 40%.
  •  
4.
  • Bergenholm, David, 1987, et al. (författare)
  • Rational gRNA design based on transcription factor binding data
  • 2021
  • Ingår i: Synthetic Biology. - : Oxford University Press (OUP). - 2397-7000 .- 1939-7267. ; 6:1
  • Tidskriftsartikel (refereegranskat)abstract
    • The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a standard tool in many genome engineering endeavors. The endonuclease-deficient version of Cas9 (dCas9) is also a powerful programmable tool for gene regulation. In this study, we made use of Saccharomyces cerevisiae transcription factor (TF) binding data to obtain a better understanding of the interplay between TF binding and binding of dCas9 fused to an activator domain, VPR. More specifically, we targeted dCas9-VPR toward binding sites of Gcr1-Gcr2 and Tye7 present in several promoters of genes encoding enzymes engaged in the central carbon metabolism. From our data, we observed an upregulation of gene expression when dCas9-VPR was targeted next to a TF binding motif, whereas a downregulation or no change was observed when dCas9 was bound on a TF motif. This suggests a steric competition between dCas9 and the specific TF. Integrating TF binding data, therefore, proved to be useful for designing guide RNAs for CRISPR interference or CRISPR activation applications.
  •  
5.
  • Bergman, Alexandra Linda, 1985, et al. (författare)
  • Effects of overexpression of STB5 in Saccharomyces cerevisiae on fatty acid biosynthesis, physiology and transcriptome
  • 2019
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 19:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Microbial conversion of biomass to fatty acids (FA) and products derived thereof is an attractive alternative to the traditional oleochemical production route from animal and plant lipids. This study examined if NADPH-costly FA biosynthesis could be enhanced by overexpressing the transcription factor Stb5 in Saccharomyces cerevisiae. Stb5 activates expression of multiple genes encoding enzymes within the pentose phosphate pathway (PPP) and other NADPH-producing reactions. Overexpression of STB5 led to a decreased growth rate and an increased free fatty acid (FFA) production during growth on glucose. The improved FFA synthetic ability in the glucose phase was shown to be independent of flux through the oxidative PPP. RNAseq analysis revealed that STB5 overexpression had wide-ranging effects on the transcriptome in the batch phase, and appeared to cause a counterintuitive phenotype with reduced flux through the oxidative PPP. During glucose limitation, when an increased NADPH supply is likely less harmful, an overall induction of the proposed target genes of Stb5 (eg. GND1/2, TAL1, ALD6, YEF1) was observed. Taken together, the strategy of utilizing STB5 overexpression to increase NADPH supply for reductive biosynthesis is suggested to have potential in strains engineered to have strong ability to consume excess NADPH, alleviating a potential redox imbalance.
  •  
6.
  • Bergman, Alexandra Linda, 1985, et al. (författare)
  • Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae
  • 2016
  • Ingår i: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 6:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Phosphoketolases catalyze an energy-and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does not demonstrate efficient phosphoketolase activity naturally. In this study, we aimed to compare and identify efficient heterologous phosphoketolase enzyme candidates that in yeast have the potential to reduce carbon loss compared to the native acetyl-CoA producing pathway by redirecting carbon flux directly from C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could largely prevent this breakdown. An evaluation of in vivo function of a subset of phosphoketolases was conducted by monitoring acetate levels during growth, confirming that candidates showing high activity in vitro indeed showed increased acetate accumulation, but expression also decreased cellular fitness. The study shows that expression of several bacterial phosphoketolase candidates in S. cerevisiae can efficiently divert intracellular carbon flux toward C2-synthesis, thus showing potential to be used in metabolic engineering strategies aimed to increase yields of acetyl-CoA derived compounds.
  •  
7.
  • Bergman, Alexandra Linda, 1985, et al. (författare)
  • Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
  • 2019
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Introduction: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized. Results: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses. Conclusion: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.
  •  
8.
  • Bergman, Alexandra Linda, 1985, et al. (författare)
  • Investigation of putative regulatory acetylation sites in Fas2p of Saccharomyces cerevisiae
  • 2018
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
    • Yeast metabolism is highly regulated, in part via coordinated reprogramming of metabolism on a transcriptional level, for example in response to environmental changes. Furthermore, regulation occurs on the protein level via posttranslational modifications directly affecting enzymatic activity – a mode of regulation that has the benefit of being very fast in response to environmental changes. One group of posttranslational modification that has been suggested to have a high impact on regulation of metabolism are acetylations. Around 4000 distinct protein acetylation sites have been found in Saccharomyces cerevisiae , many of which are located in central metabolic enzymes. However, reports on the verification of regulatory roles of specific acetylation sites on these metabolic enzymes have yet to emerge. This study investigates putative regulatory acetylation sites on Fas2p, which in concert with Fas1p is responsible for cytosolic fatty acid (FA) biosynthesis in S. cerevisiae . Fas2p stands out as one of the most highly acetylated proteins in yeast and is located at a branchpoint of acetyl-CoA metabolism. The amino acids (AAs) glutamine (Q) and arginine (R) were introduced to mimic a constitutively acetylated or non-acetylatable state at three separate lysine sites (K) (K83, K173 and K1551) confirmed to be acetylated in two independent studies, either separately or simultaneously. The results suggest that the residue replacement system in the specific case interferes with the enzymatic function of the fatty acid synthase (FAS), as QQQ and RRR triple mutants both reduce the amount of secreted free fatty acids (FFAs) in a faa1 ∆ faa4 ∆ yeast deletion mutant. The K173Q substitution significantly decreased C16 FA species at the expense of C18 FAs, while no such change could be observed for the corresponding K173R modification.
  •  
9.
  • Bergman, Alexandra Linda, 1985, et al. (författare)
  • Metabolic Engineering Strategies to Convert Carbohydrates to Aviation Range Hydrocarbons
  • 2016
  • Ingår i: Biofuels for Aviation: Feedstocks, Technology and Implementation. - 9780128045688 ; , s. 151-190
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
    • Numerous hydrocarbon molecules with properties similar to aviation fuels can be found in nature, but are often only produced at low quantities in their native hosts. This chapter summarizes recent progress in the engineering of microbial cell factories for the production of the two major molecule classes: fatty acid-derived alkanes/alkanes and isoprenoids. It starts with a brief introduction to metabolic engineering. For each of the two molecule classes, it provides information on biosynthetic pathways as well as engineering strategies to enhance their formation in the microbial hosts. While most of these endeavours are still at the proof-of concept stage, the first commercial examples are beginning to emerge.
  •  
10.
  • Blitzblau, Hannah G., et al. (författare)
  • Production of 10-methyl branched fatty acids in yeast
  • 2021
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 14:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. Results: We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. Conclusions: We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications.
  •  
11.
  • Brink, Daniel P., et al. (författare)
  • Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation
  • 2023
  • Ingår i: Microbial Cell Factories. - 1475-2859. ; 22:1
  • Tidskriftsartikel (refereegranskat)abstract
    • BACKGROUND: The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development. For instance, several strong, constitutive R. toruloides promoters have been characterized, but to date, only five inducible promoters have been identified. Although nitrogen-limited cultivation conditions are commonly used to induce lipid accumulation in this yeast, no promoters regulated by nitrogen starvation have been described for R. toruloides. RESULTS: In this study, we used a combination of genomics and transcriptomics methods to identify novel R. toruloides promoter sequences that are either inducible or repressible by nitrogen starvation. RNA sequencing was used to assess gene expression in the recently isolated strain R. toruloides BOT-A2 during exponential growth and during nitrogen starvation, when cultivated with either glucose or xylose as the carbon source. The genome of BOT-A2 was sequenced using a combination of long- and short-read sequencing and annotated with support of the RNAseq data. Differential expression analysis was used to identify genes with a |log2 fold change|≥ 2 when comparing their expression during nitrogen depletion to that during exponential growth. The promoter regions from 16 of these genes were evaluated for their ability to drive the expression of a fluorescent reporter gene. Three promoters that were clearly upregulated under nitrogen starvation and three that were downregulated were selected and further characterized. One promoter, derived from gene RTBOTA2_003877, was found to function like an on-off switch, as it was only upregulated under full nitrogen depletion and downregulated in the presence of the nitrogen source. CONCLUSIONS: Six new R. toruloides promoters that were either upregulated or downregulated under nitrogen-starvation were identified. These substantially contribute to the available promoters when engineering this organism and are foreseen to be particularly useful for future engineering strategies requiring specific regulation of target genes in accordance with nitrogen availability.
  •  
12.
  • Buijs, Nicolaas, 1985, et al. (författare)
  • Advanced biofuel production by the yeast Saccharomyces cerevisiae
  • 2013
  • Ingår i: Current Opinion in Chemical Biology. - : Elsevier BV. - 1879-0402 .- 1367-5931. ; 17:3, s. 480-488
  • Forskningsöversikt (refereegranskat)abstract
    • Replacement of conventional transportation fuels with biofuels will require production of compounds that can cover the complete fuel spectrum, ranging from gasoline to kerosene. Advanced biofuels are expected to play an important role in replacing fossil fuels because they have improved properties compared with ethanol and some of these may have the energy density required for use in heavy duty vehicles, ships, and aviation. Moreover, advanced biofuels can be used as drop-in fuels in existing internal combustion engines. The yeast cell factory Saccharomyces cerevisiae can be turned into a producer of higher alcohols (1-butanol and isobutanol), sesquiterpenes (farnesene and bisabolene), and fatty acid ethyl esters (biodiesel), and here we discusses progress in metabolic engineering of S. cerevisiae for production of these advanced biofuels.
  •  
13.
  • Buijs, Nicolaas, 1985, et al. (författare)
  • Long-chain Alkane Production by the Yeast Saccharomyces cerevisiae
  • 2015
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 112:6, s. 1275-1279
  • Tidskriftsartikel (refereegranskat)abstract
    • In the past decade industrial-scale production of renewable transportation biofuels has been developed as an alternative to fossil fuels, with ethanol as the most prominent biofuel and yeast as the production organism of choice. However, ethanol is a less efficient substitute fuel for heavy-duty and maritime transportation as well as aviation due to its low energy density. Therefore, new types of biofuels, such as alkanes, are being developed that can be used as drop-in fuels and can substitute gasoline, diesel, and kerosene. Here, we describe for the first time the heterologous biosynthesis of long-chain alkanes by the yeast Saccharomyces cerevisiae. We show that elimination of the hexadecenal dehydrogenase Hfdl and expression of a redox system are essential for alkane biosynthesis in yeast. Deletion of HFDI together with expression of an alkane biosynthesis pathway resulted in the production of the alkanes tridecane, pentadecane, and heptadecane. Our study provides a proof of principle for producing long-chain alkanes in the industrial workhorse S. cerevisiae, which was so far limited to bacteria. We anticipate that these findings will be a key factor for further yeast engineering to enable industrial production of alkane based drop-in biofuels, which can allow the biofuel industry to diversify beyond bioethanol.
  •  
14.
  • Börlin, Christoph Sebastian, 1989, et al. (författare)
  • Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions
  • 2019
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 19:2
  • Tidskriftsartikel (refereegranskat)abstract
    • One of the fundamental processes that determine cellular fate is regulation of gene transcription. Understanding these regulatory processes is therefore essential for understanding cellular responses to changes in environmental conditions. At the core promoter, the regulatory region containing the transcription start site (TSS), all inputs regulating transcription are integrated. Here, we used Cap Analysis of Gene Expression (CAGE) to analyze the pattern of TSSs at four different environmental conditions (limited in ethanol, limited in nitrogen, limited in glucose and limited in glucose under anaerobic conditions) using the Saccharomyces cerevisiae strain CEN.PK113-7D. With this experimental setup, we were able to show that the TSS landscape in yeast is stable at different metabolic states of the cell. We also show that the spatial distribution of transcription initiation events, described by the shape index, has a surprisingly strong negative correlation with measured gene expression levels, meaning that genes with higher expression levels tend to have a broader distribution of TSSs. Our analysis supplies a set of high-quality TSS annotations useful for metabolic engineering and synthetic biology approaches in the industrially relevant laboratory strain CEN.PK113-7D, and provides novel insights into yeast TSS dynamics and gene regulation.
  •  
15.
  • Börlin, Christoph Sebastian, 1989, et al. (författare)
  • The transcription factor Leu3 shows differential binding behavior in response to changing leucine availability
  • 2020
  • Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 367:13
  • Tidskriftsartikel (refereegranskat)abstract
    • The main transcriptional regulator of leucine biosynthesis in the yeast Saccharomyces cerevisiae is the transcription factor Leu3. It has previously been reported that Leu3 always binds to its target genes, but requires activation to induce their expression. In a recent large-scale study of high-resolution transcription factor binding site identification, we showed that Leu3 has divergent binding sites in different cultivation conditions, thereby questioning the results of earlier studies. Here, we present a follow-up study using chromatin immunoprecipitation followed by sequencing (ChIP-seq) to investigate the influence of leucine supplementation on Leu3 binding activity and strength. With this new data set we are able to show that Leu3 exhibits changes in binding activity in response to changing levels of leucine availability.
  •  
16.
  • Cámara, Elena, 1985, et al. (författare)
  • Saccharomyces cerevisiae strains performing similarly during fermentation of lignocellulosic hydrolysates show pronounced differences in transcriptional stress responses
  • 2024
  • Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 90:5
  • 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.
  •  
17.
  • Chao, Fang, 1983, et al. (författare)
  • A highly selective cell-based fluorescent biosensor for genistein detection
  • 2023
  • Ingår i: Engineering Microbiology. - : Elsevier BV. - 2667-3703. ; 3:2
  • Tidskriftsartikel (refereegranskat)abstract
    • Genistein, an isoflavone found mainly in legumes, has been shown to have numerous health benefits for humans. Therefore, there is substantial interest in producing it using microbial cell factories. To aid in screening for high genistein producing microbial strains, a cell-based biosensor for genistein was developed by repurposing the Gal4DBD-ERα-VP16 (GEV) transcriptional activator in Saccharomyces cerevisiae. In the presence of genistein, the GEV sensor protein binds to the GAL1 promoter and activates transcription of a downstream GFP reporter. The performance of the biosensor, as measured by fold difference in GFP signal intensity after external genistein induction, was improved by engineering the sensor protein, its promoter and the reporter promoter. Biosensor performance increased when the weak promoter REV1p was used to drive GEV sensor gene expression and the VP16 transactivating domain on GEV was replaced with the tripartite VPR transactivator that had its NLS removed. The biosensor performance further improved when the binding sites for the inhibitor Mig1 were removed from and two additional Gal4p binding sites were added to the reporter promoter. After genistein induction, our improved biosensor output a GFP signal that was 20 times higher compared to the uninduced state. Out of the 8 flavonoids tested, the improved biosensor responded only to genistein and in a somewhat linear manner. The improved biosensor also responded to genistein produced in vivo, with the GFP reporter intensity directly proportional to intracellular genistein concentration. When combined with fluorescence-based cell sorting technology, this biosensor could facilitate high-throughput screening of a genistein-producing yeast cell factory.
  •  
18.
  • Chen, Xin, 1980, et al. (författare)
  • Dataset for suppressors of amyloid-beta toxicity and their functions in recombinant protein production in yeast
  • 2022
  • Ingår i: Data in Brief. - : Elsevier BV. - 2352-3409. ; 42
  • Tidskriftsartikel (refereegranskat)abstract
    • The production of recombinant proteins at high levels often induces stress-related phenotypes by protein misfolding or aggregation. These are similar to those of the yeast Alzheimer's disease (AD) model in which amyloid-beta peptides (A beta 42) were accumulated [1,2] . We have previously identified suppressors of A beta 42 cytotoxicity via the genome-wide synthetic genetic array (SGA) [3] and here we use them as metabolic engineering targets to evaluate their potentiality on recombinant protein production in yeast Saccharomyces cerevisiae. In order to investigate the mechanisms linking the genetic modifications to the improved recombinant protein production, we perform systems biology approaches (transcriptomics and proteomics) on the resulting strain and intermediate strains. The RNAseq data are preprocessed by the nf-core/RNAseq pipeline and analyzed using the Platform for Integrative Analysis of Omics (PIANO) package [4] . The quantitative proteome is analyzed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 liquid chromatography (LC) system. LC-MS data files are processed by Proteome Discoverer version 2.4 with Mascot 2.5.1 as a database search engine. The original data presented in this work can be found in the research paper titled "Suppressors of Amyloid-beta Toxicity Improve Recombinant Protein Produc-tion in yeast by Reducing Oxidative Stress and Tuning Cellu-lar Metabolism", by Chen et al. [5] . (C) 2022 The Author(s). Published by Elsevier Inc.
  •  
19.
  • Chen, Xin, 1980, et al. (författare)
  • Suppressors of amyloid-β toxicity improve recombinant protein production in yeast by reducing oxidative stress and tuning cellular metabolism
  • 2022
  • Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 311-324
  • Tidskriftsartikel (refereegranskat)abstract
    • High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer's disease (AD) model overexpression of amyloid-β peptides (Aβ42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aβ42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins.
  •  
20.
  • Chen, Yun, 1978, et al. (författare)
  • Ach1 is involved in shuttling mitochondrial acetyl units for cytosolic C2 provision in Saccharomyces cerevisiae lacking pyruvate decarboxylase
  • 2015
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:3, s. 1-8
  • Tidskriftsartikel (refereegranskat)abstract
    • Acetyl-coenzyme A (acetyl-CoA) is not only an essential intermediate in central carbon metabolism, but also an important precursor metabolite for native or engineered pathways that can produce many products of commercial interest such as pharmaceuticals, chemicals or biofuels. In the yeast Saccharomyces cerevisiae, acetyl-CoA is compartmentalized in the cytosol, mitochondrion, peroxisome and nucleus, and cannot be directly transported between these compartments. With the acetyl-carnitine or glyoxylate shuttle, acetyl-CoA produced in peroxisomes or the cytoplasm can be transported into the cytoplasm or the mitochondria. However, whether acetyl-CoA generated in the mitochondria can be exported to the cytoplasm is still unclear. Here, we investigated whether the transfer of acetyl-CoA from the mitochondria to the cytoplasm can occur using a pyruvate decarboxylase negative, non-fermentative yeast strain. We found that mitochondrial Ach1 can convert acetyl-CoA in this compartment into acetate, which crosses the mitochondrial membrane before being converted into acetyl-CoA in the cytosol. Based on our finding we propose a model in which acetate can be used to exchange acetyl units between mitochondria and the cytosol. These results will increase our fundamental understanding of intracellular transport of acetyl units, and also help to develop microbial cell factories for many kinds of acetyl-CoA derived products.
  •  
21.
  • Chen, Yun, 1978, et al. (författare)
  • Coupled incremental precursor and co-factor supply improves 3-hydroxypropionic acid production in Saccharomyces cerevisiae
  • 2014
  • Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 22, s. 104-109
  • Tidskriftsartikel (refereegranskat)abstract
    • 3-Hydroxypropionic acid (3-HP) is an attractive platform chemical, which can be used to produce a variety of commodity chemicals, such as acrylic acid and acrylamide. For enabling a sustainable alternative to petrochemicals as the feedstock for these commercially important chemicals, fermentative production of 3-HP is widely investigated and is centered on bacterial systems in most cases. However, bacteria present certain drawbacks for large-scale organic acid production. In this study, we have evaluated the production of 3-HP in the budding yeast Saccharomyces cerevisiae through a route from malonyl-CoA, because this allows performing the fermentation at low pH thus making the overall process cheaper. We have further engineered the host strain by increasing availability of the precursor malonyl-CoA and by coupling the production with increased NADPH supply we were able to substantially improve 3-HP production by five-fold, up to a final titer of 463 mg l(-1). Our work thus led to a demonstration of 3-HP production in yeast via the malonyl-CoA pathway, and this opens for the use of yeast as a cell factory for production of bio-based 3-HP and derived acrylates in the future. (C) 2014 International Metabolic Engineering Society.
  •  
22.
  • Chen, Yun, 1978, et al. (författare)
  • Enabling Technologies to Advance Microbial Isoprenoid Production
  • 2015
  • Ingår i: Advances in Biochemical Engineering/Biotechnology. - Cham : Springer International Publishing. - 0724-6145 .- 1616-8542. ; 148, s. 143-160
  • Tidskriftsartikel (refereegranskat)abstract
    • Microbial production of isoprenoids provides an attractive alternative to biomass extraction and chemical synthesis. Although widespread research aims for isoprenoid biosynthesis, it is still in its infancy in terms of delivering commercial products. Large barriers remain in realizing a cost-competitive process, for example, developing an optimal microbial cell factory. Here, we summarize the many tools and methods that have been developed in the metabolic engineering of isoprenoid production, with the advent of systems biology and synthetic biology, and discuss how these technologies advance to accelerate the design–build–test engineering cycle to obtain optimum microbial systems. It is anticipated that innovative combinations of new and existing technologies will continue to emerge, which will enable further development of microbial cell factories for commercial isoprenoid production.
  •  
23.
  • Chen, Yun, 1978, et al. (författare)
  • Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production
  • 2012
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 12:5, s. 598-607
  • Tidskriftsartikel (refereegranskat)abstract
    • 2 mu m-based episomal expression vectors are widely used in Saccharomyces cerevisiae for recombinant protein production and synthetic pathway optimization. In this study, we report a new approach to increase the plasmid copy number (PCN) and thus improve the expression of plasmid-encoded proteins. This was achieved by combining destabilization of the marker protein with decreasing the marker gene transcription level. Destabilization of the marker protein alone by fusing a ubiquitin/N-degron tag (ubi-tag) to the N-terminus of the Ura3 marker protein could increase the PCN and activity of LacZ expressed from the same vector. When arginine was exposed at the N-terminus of the marker protein after cleavage of ubiquitin, the PCN and LacZ activity were increased by 7080%. Replacement of the native URA3 promoter with the HXT1, KEX2 or URA3-d promoter resulted in an increase in the PCN and LacZ activity by about 30100%. Combining the ubi-tag and promoter modification of the marker gene, increased the PCN and LacZ activity by threefold. We also demonstrated that this new expression vectors can be used to increase enzyme activity by improving patchoulol production by threefold.
  •  
24.
  • Chen, Yun, 1978, et al. (författare)
  • Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism
  • 2013
  • Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 15:1, s. 48-54
  • Tidskriftsartikel (refereegranskat)abstract
    • Production of fuels and chemicals by industrial biotechnology requires efficient, safe and flexible cell factory platforms that can be used for production of a wide range of compounds. Here we developed a platform yeast cell factory for efficient provision of acetyl-CoA that serves as precursor metabolite for a wide range of industrially interesting products. We demonstrate that the platform cell factory can be used to improve the production of alpha-santalene, a plant sesquiterpene that can be used as a perfume by four-fold. This strain would be a useful tool to produce a wide range of acetyl-CoA-derived products.
  •  
25.
  • Chen, Yun, 1978, et al. (författare)
  • Profiling of Cytosolic and Peroxisomal Acetyl-CoA Metabolism in Saccharomyces cerevisiae
  • 2012
  • Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 7:8, s. e42475-
  • Tidskriftsartikel (refereegranskat)abstract
    • As a key intracellular metabolite, acetyl-coenzyme A (acetyl-CoA) plays a major role in various metabolic pathways that link anabolism and catabolism. In the yeast Saccharomyces cerevisiae, acetyl-CoA involving metabolism is compartmentalized, and may vary with the nutrient supply of a cell. Membranes separating intracellular compartments are impermeable to acetyl-CoA and no direct transport between the compartments occurs. Thus, without carnitine supply the glyoxylate shunt is the sole possible route for transferring acetyl-CoA from the cytosol or the peroxisomes into the mitochondria. Here, we investigate the physiological profiling of different deletion mutants of ACS1, ACS2, CIT2 and MLS1 individually or in combination under alternative carbon sources, and study how various mutations alter carbon distribution. Based on our results a detailed model of carbon distribution about cytosolic and peroxisomal acetyl-CoA metabolism in yeast is suggested. This will be useful to further develop yeast as a cell factory for the biosynthesis of acetyl-CoA-derived products.
  •  
26.
  • Clausen Lind, Andrea, 1995, et al. (författare)
  • Evaluation and comparison of colorimetric outputs for yeast-based biosensors in laboratory and point-of-use settings
  • 2024
  • Ingår i: FEMS Microbiology Letters. - 1574-6968 .- 0378-1097. ; 371
  • Tidskriftsartikel (refereegranskat)abstract
    • Recent research has shown the potential of yeast-based biosensors (YBBs) for point-of-use detection of pathogens and target molecules in saliva, blood, and urine samples. The choice of output can greatly affect the sensitivity, dynamic range, detection time, and ease-of-use of a sensor. For visual detection without the need for additional reagents or machinery, colorimetric outputs have shown great potential. Here, we evaluated the inducible generation of prodeoxyviolacein and proviolacein as colorimetric YBB outputs and benchmarked these against lycopene. The outputs were induced via the yeast mating pathway and were compared on agar plates, in liquid culture, and on paper slips. We found that all three outputs produced comparable pigment intensity on agar plates, making them applicable for bioengineering settings. In liquid media and on paper slips, lycopene resulted in a higher intensity pigment and a decreased time-of-detection.
  •  
27.
  • Dabirian, Yasaman, 1992, et al. (författare)
  • Does co-expression of Yarrowia lipolytica genes encoding Yas1p, Yas2p and Yas3p make a potential alkane-responsive biosensor in Saccharomyces cerevisiae?
  • 2020
  • Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 15:12 December
  • Tidskriftsartikel (refereegranskat)abstract
    • Alkane-based biofuels are desirable to produce at a commercial scale as these have properties similar to current petroleum-derived transportation fuels. Rationally engineering microorganisms to produce a desirable compound, such as alkanes, is, however, challenging. Metabolic engineers are therefore increasingly implementing evolutionary engineering approaches combined with high-throughput screening tools, including metabolite biosensors, to identify productive cells. Engineering Saccharomyces cerevisiae to produce alkanes could be facilitated by using an alkane-responsive biosensor, which can potentially be developed from the native alkane-sensing system in Yarrowia lipolytica, a well-known alkaneassimilating yeast. This putative alkane-sensing system is, at least, based on three different transcription factors (TFs) named Yas1p, Yas2p and Yas3p. Although this system is not fully elucidated in Y. lipolytica, we were interested in evaluating the possibility of translating this system into an alkane-responsive biosensor in S. cerevisiae. We evaluated the alkanesensing system in S. cerevisiae by developing one sensor based on the native Y. lipolytica ALK1 promoter and one sensor based on the native S. cerevisiae CYC1 promoter. In both systems, we found that the TFs Yas1p, Yas2p and Yas3p do not seem to act in the same way as these have been reported to do in their native host. Additional analysis of the TFs suggests that more knowledge regarding their mechanism is needed before a potential alkane-responsive sensor based on the Y. lipolytica system can be established in S. cerevisiae. Copyright:
  •  
28.
  • Dabirian, Yasaman, 1992, et al. (författare)
  • Expanding the Dynamic Range of a Transcription Factor-Based Biosensor in Saccharomyces cerevisiae
  • 2019
  • Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:9, s. 1968-1975
  • Tidskriftsartikel (refereegranskat)abstract
    • Metabolite biosensors are useful tools for high-throughput screening approaches and pathway regulation approaches. An important feature of biosensors is the dynamic range. To expand the maximum dynamic range of a transcription factor-based biosensor in Saccharomyces cerevisiae, using the fapO/FapR system from Bacillus subtilis as an example case, five native promoters, including constitutive and glucose-regulated ones, were modified. By evaluating different binding site (BS) positions in the core promoters, we identified locations that resulted in a high maximum dynamic range with low expression under repressed conditions. We further identified BS positions in the upstream element region of the TEF1 promoter that did not influence the native promoter strength but resulted in repression in the presence of a chimeric repressor consisting of FapR and the yeast repressor Mig1. These modified promoters with broad dynamic ranges will provide useful information for the engineering of future biosensors and their use in complex genetic circuits.
  •  
29.
  • Dabirian, Yasaman, 1992, et al. (författare)
  • FadR-Based Biosensor-Assisted Screening for Genes Enhancing Fatty Acyl-CoA Pools in Saccharomyces cerevisiae
  • 2019
  • Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:8, s. 1788-1800
  • Tidskriftsartikel (refereegranskat)abstract
    • Fatty acid-derived compounds have a range of industrial applications, from chemical building blocks to biofuels. Due to the highly dynamic nature of fatty acid metabolism, it is difficult to identify genes modulating fatty acyl-CoA levels using a rational approach. Metabolite biosensors can be used to screen genes from large-scale libraries in vivo in a high throughput manner. Here, a fatty acyl-CoA sensor based on the transcription factor FadR from Escherichia coli was established in Saccharomyces cerevisiae and combined with a gene overexpression library to screen for genes increasing the fatty acyl-CoA pool. Fluorescence-activated cell sorting, followed by data analysis, identified genes enhancing acyl-CoA levels. From these, overexpression of RTC3, GGA2, and LPP1 resulted in about 80% increased fatty alcohol levels. Changes in fatty acid saturation and chain length distribution could also be observed. These results indicate that the use of this acyl-CoA biosensor combined with a gene overexpression library allows for identification of gene targets improving production of fatty acids and derived products.
  •  
30.
  • Dai, Zongijie, 1986, et al. (författare)
  • Global rewiring of cellular metabolism renders Saccharomyces cerevisiae Crabtree negative
  • 2018
  • Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 9:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Saccharomyces cerevisiae is a Crabtree-positive eukaryal model organism. It is believed that the Crabtree effect has evolved as a competition mechanism by allowing for rapid growth and production of ethanol at aerobic glucose excess conditions. This inherent property of yeast metabolism and the multiple mechanisms underlying it require a global rewiring of the entire metabolic network to abolish the Crabtree effect. Through rational engineering of pyruvate metabolism combined with adaptive laboratory evolution (ALE), we demonstrate that it is possible to obtain such a global rewiring and hereby turn S. cerevisiae into a Crabtree-negative yeast. Using integrated systems biology analysis, we identify that the global rewiring of cellular metabolism is accomplished through a mutation in the RNA polymerase II mediator complex, which is also observed in cancer cells expressing the Warburg effect.
  •  
31.
  • David, Florian, 1981, et al. (författare)
  • Advances in yeast genome engineering
  • 2015
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Genome engineering based on homologous recombination has been applied to yeast for many years. However, the growing importance of yeast as a cell factory in metabolic engineering and chassis in synthetic biology demands methods for fast and efficient introduction of multiple targeted changes such as gene knockouts and introduction of multistep metabolic pathways. In this review, we summarize recent improvements of existing genome engineering methods, the development of novel techniques, for example for advanced genome redesign and evolution, and the importance of endonucleases as genome engineering tools.
  •  
32.
  • David, Florian, 1981, et al. (författare)
  • Flux Control at the Malonyl-CoA Node through Hierarchical Dynamic Pathway Regulation in Saccharomyces cerevisiae
  • 2016
  • Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 5:3, s. 224-233
  • Tidskriftsartikel (refereegranskat)abstract
    • The establishment of a heterologous pathway in a microbial host for the production of industrially relevant chemicals at high titers and yields requires efficient adjustment of the central carbon metabolism to ensure that flux is directed toward the product of interest. This can be achieved through regulation at key branch points in the metabolic networks, and here we present a novel approach for dynamic modulation of pathway flux and enzyme expression levels. The approach is based on a hierarchical dynamic control system around the key pathway intermediate malonyl-CoA. The upper level of the control system ensures downregulation of endogenous use of malonyl-CoA for fatty acid biosynthesis, which results in accumulation of this pathway intermediate. The lower level of the control system is based on use of a novel biosensor for malonyl-CoA to activate expression of a heterologous pathway using this metabolite for production of 3-hydroxypropionic acid (3-HP). The malonyl-CoA sensor was developed based on the FapR transcription factor of Bacillus subtilis, and it demonstrates one of the first applications of a bacterial metabolite sensor in yeast. Introduction of the dual pathway control increased the production of 3-HP by 10-fold and can also be applied for production of other malonyl-CoA-derived products.
  •  
33.
  • de Jong, Bouke Wim, 1983, et al. (författare)
  • Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway
  • 2014
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 13:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Due to an increasing demand of transportation fuels, a lower availability of cheap crude oil and a lack of sustainability of fossil fuels, a gradual shift from petroleum based fuels towards alternative and renewable fuel resources will be required in the near future. Fatty acid ethyl esters (FAEEs) have properties similar to current crude diesel and could therefore form an important contribution to the development of sustainable transportation fuels in future. It is important to develop novel cell factories for efficient production of FAEEs and their precursors. Results: Here, a Saccharomyces cerevisiae cell factory expressing a heterologous wax ester synthase (ws2) from Marinobacter hydrocarbonoclasticus was used to produce FAEEs from ethanol and acyl-coenzyme A (acyl-CoA). The production of acyl-CoA requires large amounts of NADPH and acetyl-CoA. Therefore, two metabolic engineering strategies for improved provision of NADPH and acetyl-CoA were evaluated. First, the ethanol degradation pathway was employed to re-channel carbon flow towards the synthesis of acetyl-CoA. Therefore, ADH2 and ALD6 encoding, respectively, alcohol dehydrogenase and acetaldehyde dehydrogenase were overexpressed together with the heterologous gene acs(SE)(L641P) encoding acetyl-CoA synthetase. The co-overexpression of ADH2, ALD6 and acs(SE)(L641P) with ws2 resulted in 408 +/- 270 mu g FAEE gCDW-1, a 3-fold improvement. Secondly, for the expression of the PHK pathway two genes, xpkA and ack, both descending from Aspergillus nidulans, were co-expressed together with ws2 to catalyze, respectively, the conversion of xylulose-5-phosphate to acetyl phosphate and glyceraldehyde3-phosphate and acetyl phosphate to acetate. Alternatively, ack was substituted with pta from Bacillus subtilis, encoding phosphotransacetylase for the conversion of acetyl phosphate to acetyl-CoA. Both PHK pathways were additionally expressed in a strain with multiple chromosomally integrated ws2 gene, which resulted in respectively 5100 +/- 509 and 4670 +/- 379 mu g FAEE gCDW(-1), an up to 1.7-fold improvement. Conclusion: Two different strategies for engineering of the central carbon metabolism for efficient provision of acetyl-CoA and NADPH required for fatty acid biosynthesis and hence FAEE production were evaluated and it was found that both the ethanol degradation pathway as well as the phosphoketolase pathway improve the yield of FAEEs.
  •  
34.
  • de Jong, Bouke Wim, 1983, et al. (författare)
  • Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration
  • 2015
  • Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 42:3, s. 477-486
  • Tidskriftsartikel (refereegranskat)abstract
    • Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs(SE)(L641P)) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1(S1157A,) (S659A)) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs(SE)(L641P), ACC1(S1157A, S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1 Delta, are2 Delta, dga1 Delta and lro1 Delta) and beta-oxidation (pox1 Delta) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae.
  •  
35.
  • de Jong, Bouke Wim, 1983, et al. (författare)
  • Physiological and transcriptional characterization of Saccharomyces cerevisiae engineered for production of fatty acid ethyl esters
  • 2016
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 16:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Saccharomyces cerevisiae has previously been engineered to become a cell factory for the production of fatty acid ethyl esters (FAEEs), molecules suitable for crude diesel replacement. To find new metabolic engineering targets for the improvement of FAEE cell factories, three different FAEE-producing strains of S. cerevisiae, constructed previously, were compared and characterized by quantification of key fluxes and genome-wide transcription analysis. From both the physiological and the transcriptional data, it was indicated that strain CB2I20, with high expression of a heterologous wax ester synthase gene (ws2) and strain BdJ15, containing disruptions of genes DGA1, LRO1, ARE1, ARE2 and POX1, which prevent the conversion of acyl-CoA to sterol esters, triacylglycerides and the degradation to acetyl-CoA, triggered oxidative stress that consequently influenced cellular growth. In the latter strain, stress was possibly triggered by disabling the buffering capacity of lipid droplets in encapsulating toxic fatty acids such as oleic acid. Additionally, it was indicated that there was an increased demand for NADPH required for the reduction steps in fatty acid biosynthesis. In conclusion, our analysis clearly shows that engineering of fatty acid biosynthesis results in transcriptional reprogramming and has a significant effect on overall cellular metabolism.
  •  
36.
  • de Jong, Bouke Wim, 1983, et al. (författare)
  • Systems biology of yeast: enabling technology for development of cell factories for production of advanced biofuels
  • 2012
  • Ingår i: Current Opinion in Biotechnology. - : Elsevier BV. - 0958-1669 .- 1879-0429. ; 23:4, s. 624-630
  • Forskningsöversikt (refereegranskat)abstract
    • Transportation fuels will gradually shift from oil based fuels towards alternative fuel resources like biofuels. Current bioethanol and biodiesel can, however, not cover the increasing demand for biofuels and there is therefore a need for advanced biofuels with superior fuel properties. Novel cell factories will provide a production platform for advanced biofuels. However, deep cellular understanding is required for improvement of current biofuel cell factories. Fast screening and analysis (-omics) methods and metabolome-wide mathematical models are promising techniques. An integrated systems approach of these techniques drives diversity and quantity of several new biofuel compounds. This review will cover the recent technological developments that support improvement of the advanced biofuels 1-butanol, biodiesels and jetfuels.
  •  
37.
  • Domenzain Del Castillo Cerecer, Iván, 1991, et al. (författare)
  • Evaluating accessibility, usability and interoperability of genome-scale metabolic models for diverse yeasts species
  • 2021
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 21:1
  • Forskningsöversikt (refereegranskat)abstract
    • Metabolic network reconstructions have become an important tool for probing cellular metabolism in the field of systems biology. They are used as tools for quantitative prediction but also as scaffolds for further knowledge contextualization. The yeast Saccharomyces cerevisiae was one of the first organisms for which a genome-scale metabolic model (GEM) was reconstructed, in 2003, and since then 45 metabolic models have been developed for a wide variety of relevant yeasts species. A systematic evaluation of these models revealed that-despite this long modeling history-the sequential process of tracing model files, setting them up for basic simulation purposes and comparing them across species and even different versions, is still not a generalizable task. These findings call the yeast modeling community to comply to standard practices on model development and sharing in order to make GEMs accessible and useful for a wider public.
  •  
38.
  • Domenzain Del Castillo Cerecer, Iván, 1991, et al. (författare)
  • Reconstruction of a catalogue of genome-scale metabolic models with enzymatic constraints using GECKO 2.0
  • 2022
  • Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 13:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Genome-scale metabolic models (GEMs) have been widely used for quantitative exploration of the relation between genotype and phenotype. Streamlined integration of enzyme constraints and proteomics data into such models was first enabled by the GECKO toolbox, allowing the study of phenotypes constrained by protein limitations. Here, we upgrade the toolbox in order to enhance models with enzyme and proteomics constraints for any organism with a compatible GEM reconstruction. With this, enzyme-constrained models for the budding yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus are generated to study their long-term adaptation to several stress factors by incorporation of proteomics data. Predictions reveal that upregulation and high saturation of enzymes in amino acid metabolism are common across organisms and conditions, suggesting the relevance of metabolic robustness in contrast to optimal protein utilization as a cellular objective for microbial growth under stress and nutrient-limited conditions. The functionality of GECKO is expanded with an automated framework for continuous and version-controlled update of enzyme-constrained GEMs, also producing such models for Escherichia coli and Homo sapiens. In this work, we facilitate the utilization of enzyme-constrained GEMs in basic science, metabolic engineering and synthetic biology purposes.
  •  
39.
  • Doughty, Tyler, 1987, et al. (författare)
  • A single chromosome strain of S. cerevisiae exhibits diminished ethanol metabolism and tolerance
  • 2021
  • Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 22:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Eukaryotic organisms, like the model yeast S. cerevisiae, have linear chromosomes that facilitate organization and protection of nuclear DNA. A recent work described a stepwise break/repair method that enabled fusion of the 16 chromosomes of S. cerevisiae into a single large chromosome. Construction of this strain resulted in the removal of 30 of 32 telomeres, over 300 kb of subtelomeric DNA, and 107 subtelomeric ORFs. Despite these changes, characterization of the single chromosome strain uncovered modest phenotypes compared to a reference strain. Results: This study further characterized the single chromosome strain and found that it exhibited a longer lag phase, increased doubling time, and lower final biomass concentration compared with a reference strain when grown on YPD. These phenotypes were amplified when ethanol was added to the medium or used as the sole carbon source. RNAseq analysis showed poor induction of genes involved in diauxic shift, ethanol metabolism, and fatty-acid ß-oxidation during growth on ethanol compared to the reference strain. Enzyme-constrained metabolic modeling identified decreased flux through the enzymes that are encoded by these poorly induced genes as a likely cause of diminished biomass accumulation. The diminished growth on ethanol for the single chromosome strain was rescued by nicotinamide, an inhibitor of sirtuin family deacetylases, which have been shown to silence gene expression in heterochromatic regions. Conclusions: Our results indicate that sirtuin-mediated silencing in the single chromosome strain interferes with growth on non-fermentable carbon sources. We propose that the removal of subtelomeric DNA that would otherwise be bound by sirtuins leads to silencing at other loci in the single chromosome strain. Further, we hypothesize that the poorly induced genes in the single chromosome strain during ethanol growth could be silenced by sirtuins in wildtype S. cerevisiae during growth on glucose.
  •  
40.
  • Doughty, Tyler, 1987, et al. (författare)
  • Stress-induced expression is enriched for evolutionarily young genes in diverse budding yeasts
  • 2020
  • Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 11:1
  • Tidskriftsartikel (refereegranskat)abstract
    • The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology.
  •  
41.
  • Ferreira, Raphael, 1990, et al. (författare)
  • Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols
  • 2018
  • Ingår i: Metabolic Engineering Communications. - : Elsevier BV. - 2214-0301. ; 6, s. 22-27
  • Tidskriftsartikel (refereegranskat)abstract
    • Triacylglycerols (TAGs) are valuable versatile compounds that can be used as metabolites for nutrition and health, as well as feedstocks for biofuel production. Although Saccharomyces cerevisiae is the favored microbial cell factory for industrial production of biochemicals, it does not produce large amounts of lipids and TAGs comprise only ~1% of its cell dry weight. Here, we engineered S. cerevisiae to reorient its metabolism for overproduction of TAGs, by regulating lipid droplet associated-proteins involved in TAG synthesis and hydrolysis. We implemented a push-and-pull strategy by overexpressing genes encoding a deregulated acetyl-CoA carboxylase, ACC1 S659A/S1157A (ACC1**), as well as the last two steps of TAG formation: phosphatidic phosphatase (PAH1) and diacylglycerol acyltransferase (DGA1), ultimately leading to 129 mg∙gCDW −1 of TAGs. Disruption of TAG lipase genes TGL3, TGL4, TGL5 and sterol acyltransferase gene ARE1 increased the TAG content to 218 mg∙gCDW −1 . Further disruption of the beta-oxidation by deletion of POX1, as well as glycerol-3-phosphate utilization through deletion of GUT2, did not affect TAGs levels. Finally, disruption of the peroxisomal fatty acyl-CoA transporter PXA1 led to accumulation of 254 mg∙gCDW −1 . The TAG levels achieved here are the highest titer reported in S. cerevisiae, reaching 27.4% of the maximum theoretical yield in minimal medium with 2% glucose. This work shows the potential of using an industrially established and robust yeast species for high level lipid production.
  •  
42.
  • Ferreira, Raphael, 1990, et al. (författare)
  • Model-Assisted Fine-Tuning of Central Carbon Metabolism in Yeast through dCas9-Based Regulation
  • 2019
  • Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 8:11, s. 2457-2463
  • Tidskriftsartikel (refereegranskat)abstract
    • Engineering Saccharomyces cerevisiae for industrial-scale production of valuable chemicals involves extensive modulation of its metabolism. Here, we identified novel gene expression fine-tuning set-ups to enhance endogenous metabolic fluxes toward increasing levels of acetyl-CoA and malonyl-CoA. dCas9-based transcriptional regulation was combined together with a malonyl-CoA responsive intracellular biosensor to select for beneficial set-ups. The candidate genes for screening were predicted using a genome-scale metabolic model, and a gRNA library targeting a total of 168 selected genes was designed. After multiple rounds of fluorescence-activated cell sorting and library sequencing, the gRNAs that were functional and increased flux toward malonyl-CoA were assessed for their efficiency to enhance 3-hydroxypropionic acid (3-HP) production. 3-HP production was significantly improved upon fine-tuning genes involved in providing malonyl-CoA precursors, cofactor supply, as well as chromatin remodeling.
  •  
43.
  • Ferreira, Raphael, 1990, et al. (författare)
  • Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion
  • 2018
  • 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. ; 115:6, s. 1262-1267
  • Tidskriftsartikel (refereegranskat)abstract
    • Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in β-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8- fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories.
  •  
44.
  • Flagfeldt, D. B., et al. (författare)
  • Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae
  • 2009
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 26:10, s. 545-551
  • Tidskriftsartikel (refereegranskat)abstract
    • The construction of mitotically stable yeast strains for heterologous gene or pathway expression often requires chromosomal integration. However, transcription levels vary between different chromosome regions. We therefore characterized 20 different integration sites of the Sacchromyces cerevisiae genome by inserting lacZ as a reporter gene under the control of two different promoters and determining expression levels through enzyme activity measurement. An up to 8.7-fold difference was detected between the sites conferring lowest and highest expression, respectively. This opens the opportunity for modulating gene expression levels while retaining promoter and culture conditions. Copyright (C) 2009 John Wiley & Sons, Ltd.
  •  
45.
  • Fletcher, Eugene, 1986, et al. (författare)
  • Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments
  • 2017
  • Ingår i: Metabolic engineering. - : Academic Press. - 1096-7176 .- 1096-7184. ; 39, s. 19-28
  • Tidskriftsartikel (refereegranskat)abstract
    • Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3 M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest.
  •  
46.
  • Fletcher, Eugene, 1986, et al. (författare)
  • RNA-seq analysis of Pichia anomala reveals important mechanisms required for survival at low pH
  • 2015
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: The product yield and titers of biological processes involving the conversion of biomass to desirable chemicals can be limited by environmental stresses encountered by the microbial hosts used for the bioconversion. One of these main stresses is growth inhibition due to exposure to low pH conditions. In order to circumvent this problem, understanding the biological mechanisms involved in acid stress response and tolerance is essential. Characterisation of wild yeasts that have a natural ability to resist such harsh conditions will pave the way to understand the biological basis underlying acid stress resistance. Pichia anomala possesses a unique ability to adapt to and tolerate a number of environmental stresses particularly low pH stress giving it the advantage to outcompete other microorganisms under such conditions. However, the genetic basis of this resistance has not been previously studied. Results: To this end, we isolated an acid resistant strain of P. anomala, performed a gross phenotypic characterisation at low pH and also performed a whole genome and total RNA sequencing. By integrating the RNA-seq data with the genome sequencing data, we found that several genes associated with different biological processes including proton efflux, the electron transfer chain and oxidative phosphorylation were highly expressed in P. anomala cells grown in low pH media. We therefore present data supporting the notion that a high expression of proton pumps in the plasma membrane coupled with an increase in mitochondrial ATP production enables the high level of acid stress tolerance of P. anomala. Conclusions: Our findings provide insight into the molecular and genetic basis of low pH tolerance in P. anomala which was previously unknown. Ultimately, this is a step towards developing non-conventional yeasts such as P. anomala for the production of industrially relevant chemicals under low pH conditions.
  •  
47.
  • Gast, Veronica, 1992, et al. (författare)
  • A Hypersensitive Genetically Encoded Fluorescent Indicator (roGFP2-Prx1) Enables Continuous Measurement of Intracellular H 2 O 2 during Cell Micro-cultivation
  • 2022
  • Ingår i: Bio-protocol. - 2331-8325. ; 12:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Hydrogen peroxide (H2O2) is a toxic oxidant produced as a byproduct of several biological processes. At too high levels of hydrogen peroxide cells will experience oxidative stress, leading to a cellular response to decrease its levels and to protect the cells. Previously, methods used to study and quantify intracellular H2O2 have been limited by both sensitivity and specificity. However, an increasing number of genetically encoded fluorescent indicators (GEFIs) are becoming available, which can specifically detect low levels of intracellular hydrogen peroxide. In this study, we use such a biosensor designed to monitor cytosolic H2O2 levels in the budding yeast Saccharomyces cerevisiae during continuous cultivation and in the absence of a fluorescence microscope. The fluorescent biosensor contains a peroxiredoxin protein fused to an engineered GFP molecule expressed from a commonly used yeast plasmid (pRS416-TEF1). The peroxiredoxin-based fluorescent indicator reduces H2O2, ultimately resulting in a GFP signal being emitted by the sensor. Here, we apply this biosensor to study cytosolic H2O2 levels in S. cerevisiae strains with and without recombinant protein production.
  •  
48.
  • Gast, Veronica, 1992, et al. (författare)
  • Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules
  • 2022
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 21:1
  • Tidskriftsartikel (refereegranskat)abstract
    • BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.
  •  
49.
  • Gast, Veronica, 1992, et al. (författare)
  • The Yeast eIF2 Kinase Gcn2 Facilitates H 2 O 2 -Mediated Feedback Inhibition of Both Protein Synthesis and Endoplasmic Reticulum Oxidative Folding during Recombinant Protein Production
  • 2021
  • Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 87:15, s. e0030121-16
  • Tidskriftsartikel (refereegranskat)abstract
    • Recombinant protein production is a known source of oxidative stress. However, knowledge of which reactive oxygen species are involved or the specific growth phase in which stress occurs remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, microcultivation, and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find that Gcn2-dependent phosphorylation of eIF2α increases alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains but not in the wild type. Gcn2 deficiency furthermore reduced intracellular H2O2 levels and the Hac1 splicing ratio, while expression of antioxidants and the endoplasmic reticulum (ER) disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding are coupled and subject to feedback inhibition by H2O2. IMPORTANCE Recombinant protein production is a multibillion dollar industry. Optimizing the productivity of host cells is, therefore, of great interest. In several hosts, oxidants are produced as an unwanted side product of recombinant protein production. The buildup of oxidants can result in intracellular stress responses that could compromise the productivity of the host cell. Here, we document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (H2O2) in the cytosol of yeast cells upon the production of recombinant proteins. At the center of this inhibitory mechanism lies the protein kinase Gcn2. By removing Gcn2, we observed a doubling of recombinant protein productivity in addition to reduced H2O2 levels in the cytosol. In this study, we want to raise awareness of this inhibitory mechanism in eukaryotic cells to further improve protein production and contribute to the development of novel protein-based therapeutic strategies.
  •  
50.
  • Gibson, B., et al. (författare)
  • Adaptive Laboratory Evolution of Ale and Lager Yeasts for Improved Brewing Efficiency and Beer Quality
  • 2020
  • Ingår i: Annual review of food science and technology. - : Annual Reviews. - 1941-1421 .- 1941-1413. ; 11, s. 23-44
  • Forskningsöversikt (refereegranskat)abstract
    • Yeasts directly impact the efficiency of brewery fermentations as well as the character of the beers produced. In recent years, there has been renewed interest in yeast selection and development inspired by the demand to utilize resources more efficiently and the need to differentiate beers in a competitive market. Reviewed here are the different, non-genetically modified (GM) approaches that have been considered, including bioprospecting, hybridization, and adaptive laboratory evolution (ALE). Particular emphasis is placed on the latter, which represents an extension of the processes that have led to the domestication of strains already used in commercial breweries. ALE can be used to accentuate the positive traits of brewing yeast as well as temper some of the traits that are less desirable from a modern brewer's perspective. This method has the added advantage of being non-GM and therefore suitable for food and beverage production.
  •  
Skapa referenser, mejla, bekava och länka
  • Resultat 1-50 av 122
Typ av publikation
tidskriftsartikel (110)
forskningsöversikt (8)
bokkapitel (4)
Typ av innehåll
refereegranskat (115)
övrigt vetenskapligt/konstnärligt (7)
Författare/redaktör
Siewers, Verena, 197 ... (122)
Nielsen, Jens B, 196 ... (90)
Chen, Yun, 1978 (23)
David, Florian, 1981 (20)
Teixeira, Paulo, 199 ... (8)
Shi, Shuobo, 1981 (8)
visa fler...
Zhou, Yongjin, 1984 (7)
Li, Xiaowei, 1986 (7)
Krivoruchko, Anastas ... (7)
Ferreira, Raphael, 1 ... (6)
Engqvist, Martin, 19 ... (6)
Skrekas, Christos, 1 ... (6)
Valle Rodriguez, Jua ... (6)
Dabirian, Yasaman, 1 ... (5)
Bergman, Alexandra L ... (5)
Wenning, Leonie, 198 ... (5)
Partow, Siavash, 197 ... (5)
Scalcinati, Gionata, ... (5)
Pereira, Rui, 1986 (5)
Chen, Xin, 1980 (4)
Doughty, Tyler, 1987 (4)
Bergenholm, David, 1 ... (4)
Gossing, Michael, 19 ... (4)
Buijs, Nicolaas, 198 ... (4)
Börlin, Christoph Se ... (4)
Wang, Yanyan, 1989 (4)
Zhang, Yiming, 1986 (4)
Domenzain Del Castil ... (4)
de Jong, Bouke Wim, ... (4)
Ji, Boyang, 1983 (3)
Petranovic Nielsen, ... (3)
Molin, Mikael, 1973 (3)
Schalk, M. (3)
Hellgren, John, 1991 (3)
Nygård, Yvonne, 1986 (3)
Mormino, Maurizio, 1 ... (3)
Nookaew, Intawat, 19 ... (3)
Chao, Fang, 1983 (3)
Liu, Dany, 1994 (3)
Ishchuk, Olena, 1980 (3)
Kim, Il-Kwon, 1969 (3)
Daviet, L. (3)
Morrissey, John P. (3)
Daenen, L. (3)
Khoomrung, Sakda, 19 ... (3)
Gast, Veronica, 1992 (3)
Van Mulders, S. E. (3)
Stassen, C. (3)
van Eijsden, R. G. E ... (3)
Delvaux, F. R. (3)
visa färre...
Lärosäte
Chalmers tekniska högskola (122)
Kungliga Tekniska Högskolan (7)
Göteborgs universitet (4)
Umeå universitet (1)
Lunds universitet (1)
Sveriges Lantbruksuniversitet (1)
Språk
Engelska (122)
Forskningsämne (UKÄ/SCB)
Naturvetenskap (101)
Teknik (52)
Medicin och hälsovetenskap (23)
Lantbruksvetenskap (9)

År

Kungliga biblioteket hanterar dina personuppgifter i enlighet med EU:s dataskyddsförordning (2018), GDPR. Läs mer om hur det funkar här.
Så här hanterar KB dina uppgifter vid användning av denna tjänst.

 
pil uppåt Stäng

Kopiera och spara länken för att återkomma till aktuell vy