64 results on '"Oreb M"'
Search Results
2. Crystal Structure of Gallic Acid Decarboxylase from Arxula adeninivorans
- Author
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Zeug, M., primary, Marckovic, N., additional, Iancu, C.V., additional, Tripp, J., additional, Oreb, M., additional, and Choe, J., additional
- Published
- 2021
- Full Text
- View/download PDF
3. Crystal structure of the pea pathogenicity protein 2 from Madurella mycetomatis complexed with 4-nitrocatechol
- Author
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Zeug, M., primary, Markovic, N., additional, Iancu, C.V., additional, Tripp, J., additional, Oreb, M., additional, and Choe, J., additional
- Published
- 2021
- Full Text
- View/download PDF
4. Crystal structure of the pea pathogenicity protein 2 from Madurella mycetomatis
- Author
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Zeug, M., primary, Markovic, N., additional, Iancu, C.V., additional, Tripp, J., additional, Oreb, M., additional, and Choe, J., additional
- Published
- 2021
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- View/download PDF
5. Two‐step bioprocess for L ‐galactonate production from sugar beet pulp
- Author
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Schäfer, D., primary, Wagner, J., additional, Schmitz, K., additional, Benz, J. P., additional, Harth, S., additional, Oreb, M., additional, and Weuster-Botz, D., additional
- Published
- 2020
- Full Text
- View/download PDF
6. Engineering cofactor supply and NADH-dependent d-galacturonic acid reductases for redox-balanced production of l-galactonate in Saccharomyces cerevisiae
- Author
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Harth, S., Wagner, J., Sens, T., Choe, J., Benz, J. P., Weuster-Botz, D., and Oreb, M.
- Subjects
ddc:630 ,ddc - Published
- 2019
7. De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae
- Author
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Gottardi, M, Knudsen, J, Prado, L, Oreb, M, Branduardi, P, Boles, E, BRANDUARDI, PAOLA, Boles, E., Gottardi, M, Knudsen, J, Prado, L, Oreb, M, Branduardi, P, Boles, E, BRANDUARDI, PAOLA, and Boles, E.
- Abstract
The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.
- Published
- 2017
8. Crystal structure of Toc33 from Arabidopsis thaliana, dimerization deficient mutant R130A
- Author
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Koenig, P., primary, Oreb, M., additional, Rippe, K., additional, Muhle-Goll, C., additional, Sinning, I., additional, Schleiff, E., additional, and Tews, I., additional
- Published
- 2008
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9. De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae
- Author
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Mislav Oreb, Eckhard Boles, Jan Dines Knudsen, Paola Branduardi, Manuela Gottardi, Lydie Prado, Gottardi, M, Knudsen, J, Prado, L, Oreb, M, Branduardi, P, and Boles, E
- Subjects
0301 basic medicine ,Bioconversion ,Propanols ,Saccharomyces cerevisiae ,Arabidopsis ,Cinnamyl alcohol ,Phenylalanine ammonia-lyase ,Proof of Concept Study ,Applied Microbiology and Biotechnology ,Nocardia ,Cinnamic acid ,Cinnamaldehyde ,Metabolic engineering ,03 medical and health sciences ,chemistry.chemical_compound ,Biosynthesis ,Hydrocinnamyl alcohol ,Escherichia coli ,Acrolein ,Phenylalanine Ammonia-Lyase ,biology ,food and beverages ,General Medicine ,biology.organism_classification ,CHIM/11 - CHIMICA E BIOTECNOLOGIA DELLE FERMENTAZIONI ,Yeast ,Biosynthetic Pathways ,Glucose ,030104 developmental biology ,Metabolic Engineering ,chemistry ,Biochemistry ,Cinnamates ,trans-cinnamic acid ,Oxidoreductases ,Biotechnology - Abstract
The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.
- Published
- 2017
- Full Text
- View/download PDF
10. Engineering of Aspergillus niger for efficient production of D-xylitol from L-arabinose.
- Author
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Rüllke M, Schönrock V, Schmitz K, Oreb M, Tamayo E, and Benz JP
- Subjects
- Xylose metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae genetics, Fungal Proteins metabolism, Fungal Proteins genetics, Aspergillus niger metabolism, Aspergillus niger genetics, Arabinose metabolism, Xylitol metabolism, Xylitol biosynthesis, Metabolic Engineering methods
- Abstract
D-Xylitol is a naturally occurring sugar alcohol present in diverse plants that is used as an alternative sweetener based on a sweetness similar to sucrose and several health benefits compared to conventional sugar. However, current industrial methods for D-xylitol production are based on chemical hydrogenation of D-xylose, which is energy-intensive and environmentally harmful. However, efficient conversion of L-arabinose as an additional highly abundant pentose in lignocellulosic materials holds great potential to broaden the range of applicable feedstocks. Both pentoses D-xylose and L-arabinose are converted to D-xylitol as a common metabolic intermediate in the native fungal pentose catabolism.To engineer a strain capable of accumulating D-xylitol from arabinan-rich agricultural residues, pentose catabolism was stopped in the ascomycete filamentous fungus Aspergillus niger at the stage of D-xylitol by knocking out three genes encoding enzymes involved in D-xylitol degradation (ΔxdhA, ΔsdhA, ΔxkiA). Additionally, to facilitate its secretion into the medium, an aquaglyceroporin from Saccharomyces cerevisiae was tested. In S. cerevisiae, Fps1 is known to passively transport glycerol and is regulated to convey osmotic stress tolerance but also exhibits the ability to transport other polyols such as D-xylitol. Thus, a constitutively open version of this transporter was introduced into A. niger, controlled by multiple promoters with varying expression strengths. The strain expressing the transporter under control of the PtvdA promoter in the background of the pentose catabolism-deficient triple knock-out yielded the most favorable outcome, producing up to 45% D-xylitol from L-arabinose in culture supernatants, while displaying minimal side effects during osmotic stress. Due to its additional ability to extract D-xylose and L-arabinose from lignocellulosic material via the production of highly active pectinases and hemicellulases, A. niger emerges as an ideal candidate cell factory for D-xylitol production from lignocellulosic biomasses rich in both pentoses.In summary, we are showing for the first time an efficient biosynthesis of D-xylitol from L-arabinose utilizing a filamentous ascomycete fungus. This broadens the potential resources to include also arabinan-rich agricultural waste streams like sugar beet pulp and could thus help to make alternative sweetener production more environmentally friendly and cost-effective., (© 2024. The Author(s).)
- Published
- 2024
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11. A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study.
- Author
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Regmi P, Knesebeck M, Boles E, Weuster-Botz D, and Oreb M
- Abstract
Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae , several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L
-1 xylitol from 20 g L-1 xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase ALD6 gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP+ -dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from Kluyveromyces lactis acted synergistically with ZWF1 overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies - ZWF1 overexpression and deletion of ALD6 - also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)- Published
- 2024
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12. A yeast-based in vivo assay system for analyzing efflux of sugars mediated by glucose and xylose transporters.
- Author
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Tamayo Rojas SA, Boles E, and Oreb M
- Subjects
- Carbohydrates, Glucose metabolism, Hexokinase genetics, Hexokinase metabolism, Maltose metabolism, Monosaccharide Transport Proteins genetics, Monosaccharide Transport Proteins metabolism, Sugars metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Xylose metabolism
- Abstract
Sugar transporter research focuses on the sugar uptake into cells. Under certain physiological conditions, however, the intracellular accumulation and secretion of carbohydrates (efflux) are relevant processes in many cell types. Currently, no cell-based system is available for specifically investigating glucose efflux. Therefore, we designed a system based on a hexose transporter-deficient Saccharomyces cerevisiae strain, in which the disaccharide maltose is provided as a donor of intracellular glucose. By deleting the hexokinase genes, we prevented the metabolization of glucose, and thereby achieved the accumulation of growth-inhibitory glucose levels inside the cells. When a permease mediating glucose efflux is expressed in this system, the inhibitory effect is relieved proportionally to the capacity of the introduced transporter. The assay is thereby suitable for screening of transporters and quantitative analyses of their glucose efflux capacities. Moreover, by simultaneous provision of intracellular glucose and extracellular xylose, we investigated how each sugar influences the transport of the other one from the opposite side of the membrane. Thereby, we could show that the xylose transporter variant Gal2N376F is insensitive not only to extracellular but also to intracellular glucose. Considering the importance of sugar transporters in biotechnology, the assay could facilitate new developments in a variety of applications., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)
- Published
- 2022
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13. GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.
- Author
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Iancu CV, Bocci G, Ishtikhar M, Khamrai M, Oreb M, Oprea TI, and Choe JY
- Subjects
- Binding Sites, Biological Transport drug effects, Cell Line, Tumor, Cell Survival drug effects, Glucose Transporter Type 1 antagonists & inhibitors, Glucose Transporter Type 1 chemistry, Glucose Transporter Type 1 genetics, Glucose Transporter Type 1 metabolism, Glucose Transporter Type 2 antagonists & inhibitors, Glucose Transporter Type 2 chemistry, Glucose Transporter Type 2 genetics, Glucose Transporter Type 2 metabolism, Glucose Transporter Type 3 antagonists & inhibitors, Glucose Transporter Type 3 genetics, Glucose Transporter Type 3 metabolism, Glucose Transporter Type 4 antagonists & inhibitors, Glucose Transporter Type 4 chemistry, Glucose Transporter Type 4 genetics, Glucose Transporter Type 4 metabolism, Glucose Transporter Type 5 antagonists & inhibitors, Glucose Transporter Type 5 chemistry, Glucose Transporter Type 5 genetics, Glucose Transporter Type 5 metabolism, Heterocyclic Compounds, 3-Ring chemistry, High-Throughput Screening Assays, Humans, Models, Molecular, Neoplasms drug therapy, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins antagonists & inhibitors, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Small Molecule Libraries chemistry, Drug Discovery, Glucose Transporter Type 3 chemistry, Heterocyclic Compounds, 3-Ring pharmacology, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae Proteins chemistry, Small Molecule Libraries pharmacology
- Abstract
The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC
50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology., (© 2022. The Author(s).)- Published
- 2022
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14. Identification of a glucose-insensitive variant of Gal2 from Saccharomyces cerevisiae exhibiting a high pentose transport capacity.
- Author
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Rojas SAT, Schadeweg V, Kirchner F, Boles E, and Oreb M
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- Biological Transport, Monosaccharide Transport Proteins genetics, Mutation, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Xylose metabolism, Glucose metabolism, Monosaccharide Transport Proteins metabolism, Pentoses metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
As abundant carbohydrates in renewable feedstocks, such as pectin-rich and lignocellulosic hydrolysates, the pentoses arabinose and xylose are regarded as important substrates for production of biofuels and chemicals by engineered microbial hosts. Their efficient transport across the cellular membrane is a prerequisite for economically viable fermentation processes. Thus, there is a need for transporter variants exhibiting a high transport rate of pentoses, especially in the presence of glucose, another major constituent of biomass-based feedstocks. Here, we describe a variant of the galactose permease Gal2 from Saccharomyces cerevisiae (Gal2
N376Y/M435I ), which is fully insensitive to competitive inhibition by glucose, but, at the same time, exhibits an improved transport capacity for xylose compared to the wildtype protein. Due to this unique property, it significantly reduces the fermentation time of a diploid industrial yeast strain engineered for efficient xylose consumption in mixed glucose/xylose media. When the N376Y/M435I mutations are introduced into a Gal2 variant resistant to glucose-induced degradation, the time necessary for the complete consumption of xylose is reduced by approximately 40%. Moreover, Gal2N376Y/M435I confers improved growth of engineered yeast on arabinose. Therefore, it is a valuable addition to the toolbox necessary for valorization of complex carbohydrate mixtures., (© 2021. The Author(s).)- Published
- 2021
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15. Subcellular Localization of Fad1p in Saccharomyces cerevisiae : A Choice at Post-Transcriptional Level?
- Author
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Bruni F, Giancaspero TA, Oreb M, Tolomeo M, Leone P, Boles E, Roberti M, Caselle M, and Barile M
- Abstract
FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae , the gene encoding for FAD synthase is FAD1 , from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms-that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3' region of FAD1 mRNA was analyzed by 3'RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3'UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3'UTRs allowed us to predict the existence of a cis -acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3'UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform.
- Published
- 2021
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16. Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations.
- Author
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Wernig F, Baumann L, Boles E, and Oreb M
- Subjects
- Caprylates metabolism, Metabolic Engineering, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields., (© 2021 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals LLC.)
- Published
- 2021
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17. Identification of new GLUT2-selective inhibitors through in silico ligand screening and validation in eukaryotic expression systems.
- Author
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Schmidl S, Ursu O, Iancu CV, Oreb M, Oprea TI, and Choe JY
- Subjects
- Computer Simulation, Diabetes Mellitus drug therapy, Fanconi Syndrome drug therapy, Glucose genetics, Glucose metabolism, Glucose Transporter Type 2 chemistry, Glucose Transporter Type 2 genetics, Glucose Transporter Type 2 ultrastructure, Glucose Transporter Type 5 chemistry, Glucose Transporter Type 5 genetics, Glucose Transporter Type 5 ultrastructure, Humans, Ligands, Neoplasms drug therapy, Protein Conformation drug effects, User-Computer Interface, Drug Discovery, Glucose Transporter Type 2 antagonists & inhibitors, Glucose Transporter Type 5 antagonists & inhibitors, Small Molecule Libraries chemistry
- Abstract
Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi-Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC
50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.- Published
- 2021
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18. High-Throughput Screening of an Octanoic Acid Producer Strain Library Enables Detection of New Targets for Increasing Titers in Saccharomyces cerevisiae .
- Author
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Baumann L, Bruder S, Kabisch J, Boles E, and Oreb M
- Subjects
- Biosensing Techniques methods, Fatty Acids biosynthesis, Flow Cytometry methods, Gene Expression, Gene Library, Green Fluorescent Proteins genetics, Microorganisms, Genetically-Modified, Promoter Regions, Genetic, Saccharomyces cerevisiae genetics, Caprylates metabolism, High-Throughput Screening Assays methods, Metabolic Engineering methods, Phosphotransferases (Phosphate Group Acceptor) genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Serine Proteases genetics
- Abstract
Octanoic acid is an industrially relevant compound with applications in antimicrobials or as a precursor for biofuels. Microbial biosynthesis through yeast is a promising alternative to current unsustainable production methods. To increase octanoic acid titers in Saccharomyces cerevisiae , we use a previously developed biosensor that is based on the octanoic acid responsive pPDR12 promotor coupled to GFP. We establish a biosensor strain amenable for high-throughput screening of an octanoic acid producer strain library. Through development, optimization, and execution of a high-throughput screening approach, we were able to detect two new genetic targets, KCS1 and FSH2 , which increased octanoic acid titers through combined overexpression by about 55% compared to the parental strain. Neither target has yet been reported to be involved in fatty acid biosynthesis. The presented methodology can be employed to screen any genetic library and thereby more genes involved in improving octanoic acid production can be detected in the future.
- Published
- 2021
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19. Glucose-induced internalization of the S. cerevisiae galactose permease Gal2 is dependent on phosphorylation and ubiquitination of its aminoterminal cytoplasmic tail.
- Author
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Tamayo Rojas SA, Schmidl S, Boles E, and Oreb M
- Subjects
- Monosaccharide Transport Proteins genetics, Phosphorylation, Protein Transport, Saccharomyces cerevisiae Proteins genetics, Signal Transduction, Ubiquitin metabolism, Cytoplasm metabolism, Glucose metabolism, Monosaccharide Transport Proteins metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Ubiquitination
- Abstract
The hexose permease Gal2 of Saccharomyces cerevisiae is expressed only in the presence of its physiological substrate galactose. Glucose tightly represses the GAL2 gene and also induces the clearance of the transporter from the plasma membrane by ubiquitination and subsequent degradation in the vacuole. Although many factors involved in this process, especially those responsible for the upstream signaling, have been elucidated, the mechanisms by which Gal2 is specifically targeted by the ubiquitination machinery have remained elusive. Here, we show that ubiquitination occurs within the N-terminal cytoplasmic tail and that the arrestin-like proteins Bul1 and Rod1 are likely acting as adaptors for docking of the ubiquitin E3-ligase Rsp5. We further demonstrate that phosphorylation on multiple residues within the tail is indispensable for the internalization and possibly represents a primary signal that might trigger the recruitment of arrestins to the transporter. In addition to these new fundamental insights, we describe Gal2 mutants with improved stability in the presence of glucose, which should prove valuable for engineering yeast strains utilizing complex carbohydrate mixtures present in hydrolysates of lignocellulosic or pectin-rich biomass., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)
- Published
- 2021
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20. Transcriptomic response of Saccharomyces cerevisiae to octanoic acid production.
- Author
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Baumann L, Doughty T, Siewers V, Nielsen J, Boles E, and Oreb M
- Subjects
- Biosynthetic Pathways genetics, Caprylates analysis, Fermentation, Caprylates metabolism, Gene Expression Profiling, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- 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%., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)
- Published
- 2021
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21. Functional Expression of the Human Glucose Transporters GLUT2 and GLUT3 in Yeast Offers Novel Screening Systems for GLUT-Targeting Drugs.
- Author
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Schmidl S, Tamayo Rojas SA, Iancu CV, Choe JY, and Oreb M
- Abstract
Human GLUT2 and GLUT3, members of the GLUT/SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schmidl, Tamayo Rojas, Iancu, Choe and Oreb.)
- Published
- 2021
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22. Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists.
- Author
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Zeug M, Markovic N, Iancu CV, Tripp J, Oreb M, and Choe JY
- Abstract
Hydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids' non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5-1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid-base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.
- Published
- 2021
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23. A label-free real-time method for measuring glucose uptake kinetics in yeast.
- Author
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Schmidl S, Iancu CV, Reifenrath M, Choe JY, and Oreb M
- Subjects
- Biosensing Techniques, Glucose analysis, Green Fluorescent Proteins metabolism, Hydrogen-Ion Concentration, Kinetics, Carbohydrate Metabolism genetics, Fluorometry methods, Glucose metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism
- Abstract
Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time. The measurements are performed in the hexose transporter-deficient (hxt0) strain EBY.VW4000 that has been previously used to characterize a plethora of sugar transporters from various organisms. Therefore, this method provides an isotope-free, fluorometric approach for kinetic characterization of hexose transporters in a well-established yeast expression system., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)
- Published
- 2021
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24. Artificial ER-Derived Vesicles as Synthetic Organelles for in Vivo Compartmentalization of Biochemical Pathways.
- Author
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Reifenrath M, Oreb M, Boles E, and Tripp J
- Subjects
- Diffusion, Peptides metabolism, Proteins metabolism, Saccharomyces cerevisiae metabolism, Artificial Cells metabolism, Cytoplasmic Vesicles metabolism, Endoplasmic Reticulum metabolism, Metabolic Networks and Pathways physiology, Organelles metabolism
- Abstract
Compartmentalization in membrane-surrounded organelles has the potential to overcome obstacles associated with the engineering of metabolic pathways, such as unwanted side reactions, accumulation of toxic intermediates, drain of intermediates out of the cell, and long diffusion distances. Strategies utilizing natural organelles suffer from the presence of endogenous pathways. In our approach, we make use of endoplasmic reticulum-derived vesicles loaded with enzymes of a metabolic pathway ("metabolic vesicles"). They are generated by fusion of synthetic peptides containing the N-terminal proline-rich and self-assembling region of the maize storage protein gamma-Zein ("Zera") to the pathway enzymes. We have applied a strategy to integrate three enzymes of a cis , cis -muconic acid production pathway into those vesicles in yeast. Using fluorescence microscopy and cell fractionation techniques, we have proven the formation of metabolic vesicles and the incorporation of enzymes. Activities of the enzymes and functionality of the compartmentalized pathway were demonstrated in fermentation experiments.
- Published
- 2020
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25. Engineering cofactor supply and NADH-dependent D-galacturonic acid reductases for redox-balanced production of L-galactonate in Saccharomyces cerevisiae.
- Author
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Harth S, Wagner J, Sens T, Choe JY, Benz JP, Weuster-Botz D, and Oreb M
- Subjects
- Biotransformation, Fermentation, Oxidation-Reduction, Hexuronic Acids metabolism, NAD metabolism, NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases metabolism, Saccharomyces cerevisiae metabolism
- Abstract
D-Galacturonic acid (GalA) is the major constituent of pectin-rich biomass, an abundant and underutilized agricultural byproduct. By one reductive step catalyzed by GalA reductases, GalA is converted to the polyhydroxy acid L-galactonate (GalOA), the first intermediate of the fungal GalA catabolic pathway, which also has interesting properties for potential applications as an additive to nutrients and cosmetics. Previous attempts to establish the production of GalOA or the full GalA catabolic pathway in Saccharomyces cerevisiae proved challenging, presumably due to the inefficient supply of NADPH, the preferred cofactor of GalA reductases. Here, we tested this hypothesis by coupling the reduction of GalA to the oxidation of the sugar alcohol sorbitol that has a higher reduction state compared to glucose and thereby yields the necessary redox cofactors. By choosing a suitable sorbitol dehydrogenase, we designed yeast strains in which the sorbitol metabolism yields a "surplus" of either NADPH or NADH. By biotransformation experiments in controlled bioreactors, we demonstrate a nearly complete conversion of consumed GalA into GalOA and a highly efficient utilization of the co-substrate sorbitol in providing NADPH. Furthermore, we performed structure-guided mutagenesis of GalA reductases to change their cofactor preference from NADPH towards NADH and demonstrated their functionality by the production of GalOA in combination with the NADH-yielding sorbitol metabolism. Moreover, the engineered enzymes enabled a doubling of GalOA yields when glucose was used as a co-substrate. This significantly expands the possibilities for metabolic engineering of GalOA production and valorization of pectin-rich biomass in general.
- Published
- 2020
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26. Fusing α and β subunits of the fungal fatty acid synthase leads to improved production of fatty acids.
- Author
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Wernig F, Born S, Boles E, Grininger M, and Oreb M
- Subjects
- Artificial Gene Fusion, Fungal Proteins genetics, Gene Expression, Protein Subunits genetics, Protein Subunits metabolism, Saccharomyces cerevisiae genetics, Caprylates metabolism, Fatty Acid Synthases genetics, Fatty Acid Synthases metabolism, Fungal Proteins metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Most fungal fatty acid synthases assemble from two multidomain subunits, α and β, into a heterododecameric FAS complex. It has been recently shown that the complex assembly occurs in a cotranslational manner and is initiated by an interaction between the termini of α and β subunits. This initial engagement of subunits may be the rate-limiting phase of the assembly and subject to cellular regulation. Therefore, we hypothesized that bypassing this step by genetically fusing the subunits could be beneficial for biotechnological production of fatty acids. To test the concept, we expressed fused FAS subunits engineered for production of octanoic acid in Saccharomyces cerevisiae. Collectively, our data indicate that FAS activity is a limiting factor of fatty acid production and that FAS fusion proteins show a superior performance compared to their split counterparts. This strategy is likely a generalizable approach to optimize the production of fatty acids and derived compounds in microbial chassis organisms.
- Published
- 2020
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27. Construction of artificial membrane transport metabolons - an emerging strategy in metabolic engineering.
- Author
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Oreb M
- Subjects
- Cell Membrane metabolism, Membranes, Artificial, Metabolic Engineering trends
- Abstract
The term 'membrane transport metabolon' refers to the physical association of membrane transporters with enzymes that metabolize the transported substrates. In naturally evolved systems, physiological relevance of coupling transport with sequential enzymatic reactions resides, for instance, in faster turnover rates, protection of substrates from competing pathways or shielding the cellular environment from toxic compounds. Such underlying principles offer attractive possibilities for metabolic engineering approaches and concepts for constructing artificial transporter-enzyme complexes are recently being developed. In this minireview, the modes of substrate channeling across biological membranes and design principles for artificial transport metabolons are discussed., (© FEMS 2020.)
- Published
- 2020
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28. De novo biosynthesis of 8-hydroxyoctanoic acid via a medium-chain length specific fatty acid synthase and cytochrome P450 in Saccharomyces cerevisiae .
- Author
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Wernig F, Boles E, and Oreb M
- Abstract
Terminally hydroxylated fatty acids or dicarboxylic acids are industrially relevant compounds with broad applications. Here, we present the proof of principle for the de novo biosynthesis of 8-hydroxyoctanoic acid from glucose and ethanol in the yeast Saccharomyces cerevisiae . Toxicity tests with medium-chain length ω-hydroxy fatty acids and dicarboxylic acids revealed little or no growth impairments on yeast cultures even at higher concentrations. The ability of various heterologous cytochrome P450 enzymes in combination with their cognate reductases for ω-hydroxylation of externally fed octanoic acid were compared. Finally, the most efficient P450 enzyme system was expressed in a yeast strain, whose fatty acid synthase was engineered for octanoic acid production, resulting in de novo biosynthesis of 8-hydroxyoctanoic acid up to 3 mg/l. Accumulation of octanoic acid revealed that cytochromes P450 activities were limiting 8-hydroxyoctanoic acid synthesis. The hydroxylation of both externally added and intracellularly produced octanoic acid was strongly dependent on the carbon source used, with ethanol being preferred. We further identified the availability of heme, a cofactor needed for P450 activity, as a limiting factor of 8-hydroxyoctanoic acid biosynthesis., Competing Interests: E.B. is co-inventor of EP patent application No. 15 162 192.7 filed on April 1, 2015, and of EP patent application No. 15 174 342.4 filed on June 26, 2015, by Goethe-University Frankfurt, concerning short-chain acyl-CoA producing FAS variants. There are no other competing interests., (© 2019 The Authors.)
- Published
- 2019
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29. A Yeast-Based Biosensor for Screening of Short- and Medium-Chain Fatty Acid Production.
- Author
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Baumann L, Rajkumar AS, Morrissey JP, Boles E, and Oreb M
- Subjects
- ATP-Binding Cassette Transporters genetics, Chromatography, Gas, Fatty Acids isolation & purification, Promoter Regions, Genetic, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Biosensing Techniques methods, Fatty Acids analysis, Saccharomyces cerevisiae metabolism
- Abstract
Short- and medium-chain fatty acids (SMCFA) are important platform chemicals currently produced from nonsustainable resources. The engineering of microbial cells to produce SMCFA, however, lacks high-throughput methods to screen for best performing cells. Here, we present the development of a whole-cell biosensor for easy and rapid detection of SMCFA. The biosensor is based on a multicopy yeast plasmid containing the SMCFA-responsive PDR12 promoter coupled to GFP as the reporter gene. The sensor detected hexanoic, heptanoic and octanoic acid over a linear range up to 2, 1.5, and 0.75 mM, respectively, but did not show a linear response to decanoic and dodecanoic acid. We validated the functionality of the biosensor with culture supernatants of a previously engineered Saccharomyces cerevisiae octanoic acid producer strain and derivatives thereof. The biosensor signal correlated strongly with the octanoic acid concentrations as determined by gas chromatography. Thus, this biosensor enables the high-throughput screening of SMCFA producers and has the potential to drastically speed up the engineering of diverse SMCFA producing cell factories.
- Published
- 2018
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30. Bacterial bifunctional chorismate mutase-prephenate dehydratase PheA increases flux into the yeast phenylalanine pathway and improves mandelic acid production.
- Author
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Reifenrath M, Bauer M, Oreb M, and Boles E
- Abstract
Mandelic acid is an important aromatic fine chemical and is currently mainly produced via chemical synthesis. Recently, mandelic acid production was achieved by microbial fermentations using engineered Escherichia coli and Saccharomyces cerevisiae expressing heterologous hydroxymandelate synthases ( hmaS ). The best-performing strains carried a deletion of the gene encoding the first enzyme of the tyrosine biosynthetic pathway and therefore were auxotrophic for tyrosine. This was necessary to avoid formation of the competing intermediate hydroxyphenylpyruvate, the preferred substrate for HmaS, which would have resulted in the predominant production of hydroxymandelic acid. However, feeding tyrosine to the medium would increase fermentation costs. In order to engineer a tyrosine prototrophic mandelic acid-producing S. cerevisiae strain, we tested three strategies: (1) rational engineering of the HmaS active site for reduced binding of hydroxyphenylpyruvate, (2) compartmentalization of the mandelic acid biosynthesis pathway by relocating HmaS together with the two upstream enzymes chorismate mutase Aro7 and prephenate dehydratase Pha2 into mitochondria or peroxisomes, and (3) utilizing a feedback-resistant version of the bifunctional E. coli enzyme PheA (PheA
fbr ) in an aro7 deletion strain. PheA has both chorismate mutase and prephenate dehydratase activity. Whereas the enzyme engineering approaches were only successful in respect to reducing the preference of HmaS for hydroxyphenylpyruvate but not in increasing mandelic acid titers, we could show that strategies (2) and (3) significantly reduced hydroxymandelic acid production in favor of increased mandelic acid production, without causing tyrosine auxotrophy. Using the bifunctional enzyme PheAfbr turned out to be the most promising strategy, and mandelic acid production could be increased 12-fold, yielding titers up to 120 mg/L. Moreover, our results indicate that utilizing PheAfbr also shows promise for other industrial applications with S. cerevisiae that depend on a strong flux into the phenylalanine biosynthetic pathway.- Published
- 2018
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31. An engineered fatty acid synthase combined with a carboxylic acid reductase enables de novo production of 1-octanol in Saccharomyces cerevisiae .
- Author
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Henritzi S, Fischer M, Grininger M, Oreb M, and Boles E
- Abstract
Background: The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway., Results: The previously engineered short-chain acyl-CoA producing yeast Fas1
R1834K /Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L-1 in a 72-h fermentation. The additional accumulation of 90 mg L-1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L-1 . However, in growth tests concentrations even lower than 50.0 mg L-1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake., Conclusions: By providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae . Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.- Published
- 2018
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32. Ligand Screening Systems for Human Glucose Transporters as Tools in Drug Discovery.
- Author
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Schmidl S, Iancu CV, Choe JY, and Oreb M
- Abstract
Hexoses are the major source of energy and carbon skeletons for biosynthetic processes in all kingdoms of life. Their cellular uptake is mediated by specialized transporters, including glucose transporters (GLUT, SLC2 gene family). Malfunction or altered expression pattern of GLUTs in humans is associated with several widespread diseases including cancer, diabetes and severe metabolic disorders. Their high relevance in the medical area makes these transporters valuable drug targets and potential biomarkers. Nevertheless, the lack of a suitable high-throughput screening system has impeded the determination of compounds that would enable specific manipulation of GLUTs so far. Availability of structural data on several GLUTs enabled in silico ligand screening, though limited by the fact that only two major conformations of the transporters can be tested. Recently, convenient high-throughput microbial and cell-free screening systems have been developed. These remarkable achievements set the foundation for further and detailed elucidation of the molecular mechanisms of glucose transport and will also lead to great progress in the discovery of GLUT effectors as therapeutic agents. In this mini-review, we focus on recent efforts to identify potential GLUT-targeting drugs, based on a combination of structural biology and different assay systems.
- Published
- 2018
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33. An expanded enzyme toolbox for production of cis, cis-muconic acid and other shikimate pathway derivatives in Saccharomyces cerevisiae.
- Author
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Brückner C, Oreb M, Kunze G, Boles E, and Tripp J
- Subjects
- Gene Expression, Gene Expression Regulation, Fungal, Mutation, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Shikimic Acid chemistry, Sorbic Acid chemistry, Sorbic Acid metabolism, Metabolic Networks and Pathways, Saccharomyces cerevisiae metabolism, Shikimic Acid metabolism, Sorbic Acid analogs & derivatives
- Abstract
A wide range of commercially relevant aromatic chemicals can be synthesized via the shikimic acid pathway. Thus, this pathway has been the target of diverse metabolic engineering strategies. In the present work, an optimized yeast strain for production of the shikimic acid pathway intermediate 3-dehydroshikimate (3-DHS) was generated, which is a precursor for the production of the valuable compounds cis, cis-muconic acid (CCM) and gallic acid (GA). Production of CCM requires the overexpression of the heterologous enzymes 3-DHS dehydratase AroZ, protocatechuic acid (PCA) decarboxylase AroY and catechol dioxygenase CatA. The activity of AroY limits the yield of the pathway. This repertoire of enzymes was expanded by a novel fungal decarboxylase. Introducing this enzyme into the pathway in the optimized strain, a titer of 1244 mg L-1 CCM could be achieved, yielding 31 mg g-1 glucose. This represents the highest yield of this compound reported in Saccharomyces cerevisiae to date. To demonstrate the applicability of the optimized strain for production of other compounds from 3-DHS, we overexpressed AroZ together with a mutant of a para-hydroxybenzoic acid hydroxylase with improved substrate specificity for PCA, PobAY385F. Thereby, we could demonstrate the production of GA for the first time in S. cerevisiae.
- Published
- 2018
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34. A Growth-Based Screening System for Hexose Transporters in Yeast.
- Author
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Boles E and Oreb M
- Subjects
- Biological Transport drug effects, Cloning, Molecular, Drug Discovery methods, Gene Expression, Genetic Complementation Test, Monosaccharide Transport Proteins antagonists & inhibitors, Monosaccharide Transport Proteins genetics, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Biological Assay, Monosaccharide Transport Proteins metabolism, Sugars metabolism
- Abstract
As the simplest eukaryotic model system, the unicellular yeast Saccharomyces cerevisiae is ideally suited for quick and simple functional studies as well as for high-throughput screening. We generated a strain deficient for all endogenous hexose transporters, which has been successfully used to clone, characterize, and engineer carbohydrate transporters from different source organisms. Here we present basic protocols for handling this strain and characterizing sugar transporters heterologously expressed in it.
- Published
- 2018
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35. Optimisation of trans-cinnamic acid and hydrocinnamyl alcohol production with recombinant Saccharomyces cerevisiae and identification of cinnamyl methyl ketone as a by-product.
- Author
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Gottardi M, Grün P, Bode HB, Hoffmann T, Schwab W, Oreb M, and Boles E
- Subjects
- Alcohols chemistry, Chromatography, High Pressure Liquid, Cinnamates chemistry, Fermentation, Genes, Plant, Glucose biosynthesis, Mass Spectrometry, Metabolic Networks and Pathways, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Alcohols metabolism, Cinnamates metabolism, Ketones metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Trans-cinnamic acid (tCA) and hydrocinnamyl alcohol (HcinOH) are valuable aromatic compounds with applications in the flavour, fragrance and cosmetic industry. They can be produced with recombinant yeasts from sugars via phenylalanine after expression of a phenylalanine ammonia lyase (PAL) and an aryl carboxylic acid reductase. Here, we show that in Saccharomyces cerevisiae a PAL enzyme from the bacterium Photorhabdus luminescens was superior to a previously used plant PAL enzyme for the production of tCA. Moreover, after expression of a UDP-glucose:cinnamate glucosyltransferase (FaGT2) from Fragaria x ananassa, tCA could be converted to cinnamoyl-D-glucose which is expected to be less toxic to the yeast cells. Production of tCA and HcinOH from glucose could be increased by eliminating feedback-regulated steps of aromatic amino acid biosynthesis and diminishing the decarboxylation step of the competing Ehrlich pathway. Finally, an unknown by-product resulting from further metabolisation of a carboligation product of cinnamaldehyde (cinALD) with activated acetaldehyde, mediated by pyruvate decarboxylases, could be identified as cinnamyl methyl ketone providing a new route for the biosynthesis of precursors, such as (2S,3R) 5-phenylpent-4-ene-2,3-diol, necessary for the chemical synthesis of specific biologically active drugs such as daunomycin., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2017
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36. An artificial transport metabolon facilitates improved substrate utilization in yeast.
- Author
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Thomik T, Wittig I, Choe JY, Boles E, and Oreb M
- Subjects
- Biofuels, Biological Transport, Fermentation, Metabolomics, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae growth & development, Substrate Specificity, Xylitol metabolism, Aldose-Ketose Isomerases metabolism, Ethanol metabolism, Saccharomyces cerevisiae metabolism, Xylose metabolism
- Abstract
Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.
- Published
- 2017
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37. Establishing a yeast-based screening system for discovery of human GLUT5 inhibitors and activators.
- Author
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Tripp J, Essl C, Iancu CV, Boles E, Choe JY, and Oreb M
- Subjects
- Biological Transport, Catechin analogs & derivatives, Catechin pharmacology, Drug Evaluation, Preclinical, Enzyme Inhibitors pharmacology, Glucose Transporter Type 5 antagonists & inhibitors, Glucose Transporter Type 5 chemistry, Glucose Transporter Type 5 genetics, High-Throughput Screening Assays, Humans, Kinetics, Ligands, Models, Molecular, Protein Conformation, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Enzyme Inhibitors isolation & purification, Fructose metabolism, Glucose Transporter Type 5 metabolism, Mutation
- Abstract
Human GLUT5 is a fructose-specific transporter in the glucose transporter family (GLUT, SLC2 gene family). Its substrate-specificity and tissue-specific expression make it a promising target for treatment of diabetes, metabolic syndrome and cancer, but few GLUT5 inhibitors are known. To identify and characterize potential GLUT5 ligands, we developed a whole-cell system based on a yeast strain deficient in fructose uptake, in which GLUT5 transport activity is associated with cell growth in fructose-based media or assayed by fructose uptake in whole cells. The former method is convenient for high-throughput screening of potential GLUT5 inhibitors and activators, while the latter enables detailed kinetic characterization of identified GLUT5 ligands. We show that functional expression of GLUT5 in yeast requires mutations at specific positions of the transporter sequence. The mutated proteins exhibit kinetic properties similar to the wild-type transporter and are inhibited by established GLUT5 inhibitors N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) and (-)-epicatechin-gallate (ECG). Thus, this system has the potential to greatly accelerate the discovery of compounds that modulate the fructose transport activity of GLUT5.
- Published
- 2017
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38. De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.
- Author
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Gottardi M, Knudsen JD, Prado L, Oreb M, Branduardi P, and Boles E
- Subjects
- Acrolein analogs & derivatives, Acrolein metabolism, Arabidopsis enzymology, Arabidopsis genetics, Biosynthetic Pathways, Cinnamates chemistry, Escherichia coli enzymology, Escherichia coli genetics, Glucose metabolism, Nocardia enzymology, Nocardia genetics, Oxidoreductases genetics, Phenylalanine Ammonia-Lyase genetics, Proof of Concept Study, Propanols metabolism, Cinnamates metabolism, Metabolic Engineering methods, Saccharomyces cerevisiae metabolism
- Abstract
The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.
- Published
- 2017
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39. Secretion of 2,3-dihydroxyisovalerate as a limiting factor for isobutanol production in Saccharomyces cerevisiae.
- Author
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Generoso WC, Brinek M, Dietz H, Oreb M, and Boles E
- Subjects
- Biofuels, Butyrates antagonists & inhibitors, Fermentation, Industrial Microbiology, Kinetics, Microarray Analysis, Saccharomyces cerevisiae metabolism, Valerates, Butanols metabolism, Butyrates metabolism, Gene Deletion, Genes, Fungal, Genetic Engineering methods, Saccharomyces cerevisiae genetics
- Abstract
Isobutanol is a superior biofuel compared to ethanol, and it is naturally produced by yeasts. Previously, Saccharomyces cerevisiae has been genetically engineered to improve isobutanol production. We found that yeast cells engineered for a cytosolic isobutanol biosynthesis secrete large amounts of the intermediate 2,3-dihydroxyisovalerate (DIV). This indicates that the enzyme dihydroxyacid dehydratase (Ilv3) is limiting the isobutanol pathway and/or yeast exhibit effective transport systems for the secretion of the intermediate, competing with isobutanol synthesis. Moreover, we found that DIV cannot be taken up by the cells again. To identify the responsible transporters, microarray analysis was performed with a DIV producing strain compared to a wild type. Altogether, 19 genes encoding putative transporters were upregulated under DIV-producing conditions. Thirteen of these were deleted together with five homologous genes. A yro2 mrh1 deletion strain showed reduced DIV secretion, while a hxt5 deletion mutant showed increased isobutanol production. However, a strain deleted for all the 18 genes secreted even slightly increased amounts of the intermediates and less isobutanol. The lactate transporter Jen1 turned out to transport the intermediate 2-ketoisovalerate, but not DIV. The results suggest that the transport of DIV is a rather complex process and several unspecific transporters seem to be involved., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2017
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40. Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae.
- Author
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Weber HE, Gottardi M, Brückner C, Oreb M, Boles E, and Tripp J
- Subjects
- Bacterial Proteins genetics, Carboxy-Lyases genetics, Dimethylallyltranstransferase genetics, Hydroxybenzoates metabolism, Klebsiella pneumoniae enzymology, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Sorbic Acid metabolism, Bacterial Proteins metabolism, Carboxy-Lyases metabolism, Dimethylallyltranstransferase metabolism, Flavin Mononucleotide metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Sorbic Acid analogs & derivatives
- Abstract
Biotechnological production of cis , cis -muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis , cis -muconic acid has already been established in the host organism Saccharomyces cerevisiae , the generation of industrially relevant amounts of cis , cis -muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit C
iso (AroY-Ciso ), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-Ciso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1 , which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-Ciso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-Ciso , although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-Ciso , suggesting that mitochondrial localization has no major impact on cofactor biosynthesis. IMPORTANCE In Saccharomyces cerevisiae , the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis , cis -muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain., (Copyright © 2017 American Society for Microbiology.)- Published
- 2017
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41. Polymorphisms in the LAC12 gene explain lactose utilisation variability in Kluyveromyces marxianus strains.
- Author
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Varela JA, Montini N, Scully D, Van der Ploeg R, Oreb M, Boles E, Hirota J, Akada R, Hoshida H, and Morrissey JP
- Subjects
- Amino Acid Sequence, Chromosome Mapping, Chromosomes, Fungal chemistry, Culture Media chemistry, Fermentation, Fungal Proteins metabolism, Gene Dosage, Kinetics, Kluyveromyces classification, Kluyveromyces enzymology, Membrane Transport Proteins metabolism, Phylogeny, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Sequence Alignment, Sequence Homology, Amino Acid, Substrate Specificity, Fungal Proteins genetics, Gene Expression Regulation, Fungal, Kluyveromyces genetics, Lactose metabolism, Membrane Transport Proteins genetics, Polymorphism, Genetic
- Abstract
Kluyveromyces marxianus is a safe yeast used in the food and biotechnology sectors. One of the important traits that sets it apart from the familiar yeasts, Saccharomyces cerevisiae, is its capacity to grow using lactose as a carbon source. Like in its close relative, Kluyveromyces lactis, this requires lactose transport via a permease and intracellular hydrolysis of the disaccharide. Given the importance of the trait, it was intriguing that most, but not all, strains of K. marxianus are reported to consume lactose efficiently. In this study, primarily through heterologous expression in S. cerevisiae and K. marxianus, it was established that a single gene, LAC12, is responsible for lactose uptake in K. marxianus. Strains that failed to transport lactose showed variation in 13 amino acids in the Lac12p protein, rendering the protein non-functional for lactose transport. Genome analysis showed that the LAC12 gene is present in four copies in the subtelomeric regions of three different chromosomes but only the ancestral LAC12 gene encodes a functional lactose transporter. Other copies of LAC12 may be non-functional or have alternative substrates. The analysis raises some interesting questions regarding the evolution of sugar transporters in K. marxianus., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2017
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42. Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae.
- Author
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Generoso WC, Gottardi M, Oreb M, and Boles E
- Subjects
- Gene Deletion, CRISPR-Cas Systems, Gene Editing methods, Genetic Engineering methods, Genome, Fungal, Saccharomyces cerevisiae genetics
- Abstract
CRISPR-Cas has become a powerful technique for genetic engineering of yeast. Here, we present an improved version by using only one single plasmid expressing Cas9 and one or two guide-RNAs. A high gene deletion efficiency was achieved even with simultaneous recombination cloning of the plasmid and deletion in industrial strains., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
43. Hxt13, Hxt15, Hxt16 and Hxt17 from Saccharomyces cerevisiae represent a novel type of polyol transporters.
- Author
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Jordan P, Choe JY, Boles E, and Oreb M
- Subjects
- Membrane Transport Proteins chemistry, Molecular Docking Simulation, Monosaccharide Transport Proteins chemistry, Saccharomyces cerevisiae Proteins chemistry, L-Iditol 2-Dehydrogenase metabolism, Mannitol metabolism, Membrane Transport Proteins metabolism, Monosaccharide Transport Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism, Sorbitol metabolism
- Abstract
The genome of S. cerevisae encodes at least twenty hexose transporter-like proteins. Despite extensive research, the functions of Hxt8-Hxt17 have remained poorly defined. Here, we show that Hxt13, Hxt15, Hxt16 and Hxt17 transport two major hexitols in nature, mannitol and sorbitol, with moderate affinities, by a facilitative mechanism. Moreover, Hxt11 and Hxt15 are capable of transporting xylitol, a five-carbon polyol derived from xylose, the most abundant pentose in lignocellulosic biomass. Hxt11, Hxt13, Hxt15, Hxt16 and Hxt17 are phylogenetically and functionally distinct from known polyol transporters. Based on docking of polyols to homology models of transporters, we propose the architecture of their active site. In addition, we determined the kinetic parameters of mannitol and sorbitol dehydrogenases encoded in the yeast genome, showing that they discriminate between mannitol and sorbitol to a much higher degree than the transporters.
- Published
- 2016
- Full Text
- View/download PDF
44. Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers.
- Author
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Generoso WC, Schadeweg V, Oreb M, and Boles E
- Subjects
- 1-Butanol chemistry, Biomass, Butanols chemistry, Isomerism, Saccharomyces cerevisiae genetics, 1-Butanol metabolism, Butanols metabolism, Metabolic Engineering, Saccharomyces cerevisiae metabolism
- Abstract
Saccharomyces cerevisiae has decisive advantages in industrial processes due to its tolerance to alcohols and fermentation conditions. Butanol isomers are considered as suitable fuel substitutes and valuable biomass-derived chemical building blocks. Whereas high production was achieved with bacterial systems, metabolic engineering of yeast for butanol production is in the beginning. For isobutanol synthesis, combination of valine biosynthesis and degradation, and complete pathway re-localisation into cytosol or mitochondria gave promising results. However, competing pathways, co-factor imbalances and FeS cluster assembly are still major issues. 1-Butanol production via the Clostridium pathway seems to be limited by cytosolic acetyl-CoA, its central precursor. Endogenous 1-butanol pathways have been discovered via threonine or glycine catabolism. 2-Butanol production was established but was limited by B12-dependence., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
45. On the role of GAPDH isoenzymes during pentose fermentation in engineered Saccharomyces cerevisiae.
- Author
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Linck A, Vu XK, Essl C, Hiesl C, Boles E, and Oreb M
- Subjects
- Fermentation, Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) genetics, Isoenzymes genetics, Isoenzymes metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Transaldolase metabolism, Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) metabolism, Metabolic Engineering, Pentoses metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
In the metabolic network of the cell, many intermediary products are shared between different pathways. d-Glyceraldehyde-3-phosphate, a glycolytic intermediate, is a substrate of GAPDH but is also utilized by transaldolase and transketolase in the scrambling reactions of the nonoxidative pentose phosphate pathway. Recent efforts to engineer baker's yeast strains capable of utilizing pentose sugars present in plant biomass rely on increasing the carbon flux through this pathway. However, the competition between transaldolase and GAPDH for d-glyceraldehyde-3-phosphate produced in the first transketolase reaction compromises the carbon balance of the pathway, thereby limiting the product yield. Guided by the hypothesis that reduction in GAPDH activity would increase the availability of d-glyceraldehyde-3-phosphate for transaldolase and thereby improve ethanol production during fermentation of pentoses, we performed a comprehensive characterization of the three GAPDH isoenzymes in baker's yeast, Tdh1, Tdh2, and Tdh3 and analyzed the effect of their deletion on xylose utilization by engineered strains. Our data suggest that overexpression of transaldolase is a more promising strategy than reduction in GAPDH activity to increase the flux through the nonoxidative pentose phosphate pathway., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
46. Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose.
- Author
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Farwick A, Bruder S, Schadeweg V, Oreb M, and Boles E
- Subjects
- Biological Transport, Hydrolysis, Models, Molecular, Monosaccharide Transport Proteins antagonists & inhibitors, Monosaccharide Transport Proteins chemistry, Monosaccharide Transport Proteins genetics, Mutagenesis, Site-Directed, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae growth & development, Glucose metabolism, Monosaccharide Transport Proteins metabolism, Protein Engineering, Saccharomyces cerevisiae metabolism, Xylose metabolism
- Abstract
All known D-xylose transporters are competitively inhibited by D-glucose, which is one of the major reasons hampering simultaneous fermentation of D-glucose and D-xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growth-based screening system for mutant D-xylose transporters that are insensitive to the presence of D-glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the D-xylose transporter XylE from Escherichia coli, both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent D-glucose but not D-xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of D-glucose negatively affected transport of both D-glucose and D-xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for D-xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose-xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.
- Published
- 2014
- Full Text
- View/download PDF
47. Nucleotides and substrates trigger the dynamics of the Toc34 GTPase homodimer involved in chloroplast preprotein translocation.
- Author
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Lumme C, Altan-Martin H, Dastvan R, Sommer MS, Oreb M, Schuetz D, Hellenkamp B, Mirus O, Kretschmer J, Lyubenova S, Kügel W, Medelnik JP, Dehmer M, Michaelis J, Prisner TF, Hugel T, and Schleiff E
- Subjects
- Amino Acid Sequence, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Kinetics, Membrane Proteins genetics, Models, Molecular, Molecular Sequence Data, Plant Proteins genetics, Protein Binding, Protein Multimerization, Protein Precursors genetics, Protein Transport, Recombinant Proteins chemistry, Recombinant Proteins genetics, Substrate Specificity, Thermodynamics, Chloroplasts chemistry, Guanosine Diphosphate chemistry, Guanosine Triphosphate chemistry, Membrane Proteins chemistry, Pisum sativum chemistry, Plant Proteins chemistry, Protein Precursors chemistry
- Abstract
GTPases are molecular switches that control numerous crucial cellular processes. Unlike bona fide GTPases, which are regulated by intramolecular structural transitions, the less well studied GAD-GTPases are activated by nucleotide-dependent dimerization. A member of this family is the translocase of the outer envelope membrane of chloroplast Toc34 involved in regulation of preprotein import. The GTPase cycle of Toc34 is considered a major circuit of translocation regulation. Contrary to expectations, previous studies yielded only marginal structural changes of dimeric Toc34 in response to different nucleotide loads. Referencing PELDOR and FRET single-molecule and bulk experiments, we describe a nucleotide-dependent transition of the dimer flexibility from a tight GDP- to a flexible GTP-loaded state. Substrate binding induces an opening of the GDP-loaded dimer. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which likely represents a general regulatory mode for dimerizing GTPases., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
48. Novel strategies to improve co-fermentation of pentoses with D-glucose by recombinant yeast strains in lignocellulosic hydrolysates.
- Author
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Oreb M, Dietz H, Farwick A, and Boles E
- Subjects
- Bacterial Proteins metabolism, Biofuels, Fermentation, Genetic Engineering, Glucose metabolism, Hydrolysis, Monosaccharide Transport Proteins metabolism, Saccharomyces cerevisiae genetics, Transgenes, Bacterial Proteins genetics, Ethanol metabolism, Lignin metabolism, Monosaccharide Transport Proteins genetics, Pentoses metabolism, Saccharomyces cerevisiae metabolism
- Abstract
Economically feasible production of second-generation biofuels requires efficient co-fermentation of pentose and hexose sugars in lignocellulosic hydrolysates under very harsh conditions. Baker's yeast is an excellent, traditionally used ethanol producer but is naturally not able to utilize pentoses. This is due to the lack of pentose-specific transporter proteins and enzymatic reactions. Thus, natural yeast strains must be modified by genetic engineering. Although the construction of various recombinant yeast strains able to ferment pentose sugars has been described during the last two decades, their rates of pentose utilization is still significantly lower than D-glucose fermentation. Moreover, pentoses are only fermented after D-glucose is exhausted, resulting in an uneconomical increase in the fermentation time. In this addendum, we discuss novel approaches to improve utilization of pentoses by development of specific transporters and substrate channeling in enzyme cascades.
- Published
- 2012
- Full Text
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49. Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33.
- Author
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Oreb M, Höfle A, Koenig P, Sommer MS, Sinning I, Wang F, Tews I, Schnell DJ, and Schleiff E
- Subjects
- Amino Acid Sequence, Arabidopsis Proteins genetics, Chloroplasts genetics, GTP Phosphohydrolases genetics, Guanosine Diphosphate genetics, Guanosine Triphosphate genetics, Membrane Proteins genetics, Molecular Sequence Data, Protein Binding genetics, Protein Multimerization genetics, Protein Precursors metabolism, Substrate Specificity genetics, Arabidopsis Proteins metabolism, Chloroplasts enzymology, GTP Phosphohydrolases metabolism, Guanosine Diphosphate metabolism, Guanosine Triphosphate metabolism, Membrane Proteins metabolism, Protein Multimerization physiology
- Abstract
GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast.
- Published
- 2011
- Full Text
- View/download PDF
50. pH sensitivity of the GTPase Toc33 as a regulatory circuit for protein translocation into chloroplasts.
- Author
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Bionda T, Koenig P, Oreb M, Tews I, and Schleiff E
- Subjects
- Hydrogen-Ion Concentration, Protein Binding, Protein Multimerization, Protein Precursors metabolism, Protein Transport, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Chloroplasts metabolism, GTP Phosphohydrolases metabolism, Membrane Proteins metabolism
- Abstract
The properties of membrane-embedded GTPases are investigated to understand translocation of preprotein across the outer envelope of chloroplasts. The homo- and heterodimerization events of the GTPases had been established previously. We show that the hydrolytic activity of the GTPase Toc33 is pH insensitive in the homodimeric conformation but has a bell-shaped pH optimum in the monomeric conformation. Further, Toc33 GTPase homodimerization and protein translocation into chloroplasts are pH sensitive as well. pH sensitivity might serve to regulate translocation; alternatively, the documented pH sensitivity might reflect a mechanistic requirement for GTPase silencing during translocation as the GTPase switches between homo- and heterodimeric conformations.
- Published
- 2008
- Full Text
- View/download PDF
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