scholarly journals Cofactor Dependence in Furan Reduction by Saccharomyces cerevisiae in Fermentation of Acid-Hydrolyzed Lignocellulose

2005 ◽  
Vol 71 (12) ◽  
pp. 7866-7871 ◽  
Author(s):  
Anneli Nilsson ◽  
Marie F. Gorwa-Grauslund ◽  
Bärbel Hahn-Hägerdal ◽  
Gunnar Lidén
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Degradation Products ◽  
Fed Batch ◽  
Fermentation Rate ◽  
Tolerant Strain ◽  
Acid Hydrolysate ◽  
Hydroxymethyl Furfural ◽  
Cell Extracts ◽  
Sugar Degradation

ABSTRACT A decreased fermentation rate due to inhibition is a significant problem for economic conversion of acid-pretreated lignocellulose hydrolysates to ethanol, since the inhibition gives rise to a requirement for separate detoxification steps. Together with acetic acid, the sugar degradation products furfural and 5-hydroxymethyl furfural are the inhibiting compounds found at the highest concentrations in hydrolysates. These aldehydes have been shown to affect both the specific growth rate and the rate of fermentation by yeast. Two strains of Saccharomyces cerevisiae with different abilities to ferment inhibiting hydrolysates were evaluated in fermentations of a dilute acid hydrolysate from spruce, and the reducing activities for furfural and 5-hydroxymethyl furfural were determined. Crude cell extracts of a hydrolysate-tolerant strain (TMB3000) converted both furfural and 5-hydroxymethyl furfural to the corresponding alcohol at a rate that was severalfold higher than the rate observed for cell extracts of a less tolerant strain (CBS 8066), thereby confirming that there is a correlation between the fermentation rate in a lignocellulosic hydrolysate and the bioconversion capacity of a strain. The in vitro NADH-dependent furfural reduction capacity of TMB3000 was three times higher than that of CBS 8066 (1,200 mU/mg protein and 370 mU/mg protein, respectively) in fed-batch experiments. Furthermore, the inhibitor-tolerant strain TMB3000 displayed a previously unknown NADH-dependent reducing activity for 5-hydroxymethyl furfural (400 mU/mg protein during fed-batch fermentation of hydrolysates). No corresponding activity was found in strain CBS 8066 (<2 mU/mg). The ability to reduce 5-hydroxymethyl furfural is an important characteristic for the development of yeast strains with increased tolerance to lignocellulosic hydrolysates.

Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase

1986 ◽  
Vol 6 (7) ◽  
pp. 2382-2391
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Mutant Gene ◽  
Yeast Cells ◽  
Molecular Weight Characteristic ◽  
Cell Extracts ◽  
Substitution Mutations ◽  
Membrane Components

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

scholarly journals Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase.

1986 ◽  
Vol 6 (7) ◽  
pp. 2382-2391 ◽  
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Mutant Gene ◽  
Yeast Cells ◽  
Molecular Weight Characteristic ◽  
Cell Extracts ◽  
Substitution Mutations ◽  
Membrane Components

Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro. The consequences of these mutations were studied after returning the mutated genes to yeast cells. Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase. Other substitution mutations and longer deletions blocked the formation of extracellular invertase. Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties. The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified. The large increase in molecular weight characteristic of glycosylation was not seen. On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme. All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients. Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway. This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

scholarly journals Prion Filament Networks in [Ure3] Cells of Saccharomyces cerevisiae

2001 ◽  
Vol 153 (6) ◽  
pp. 1327-1336 ◽  
Author(s):  
Vladislav V. Speransky ◽  
Kimberly L. Taylor ◽  
Herman K. Edskes ◽  
Reed B. Wickner ◽  
Alasdair C. Steven
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Normal Function ◽  
Nitrogen Regulation ◽  
Wild Type ◽  
Cell Extracts ◽  
Cytoplasmic Filaments ◽  
Altered Form ◽  
Filament Networks

The [URE3] prion (infectious protein) of yeast is a self-propagating, altered form of Ure2p that cannot carry out its normal function in nitrogen regulation. Previous data have shown that Ure2p can form protease-resistant amyloid filaments in vitro, and that it is aggregated in cells carrying the [URE3] prion. Here we show by electron microscopy that [URE3] cells overexpressing Ure2p contain distinctive, filamentous networks in their cytoplasm, and demonstrate by immunolabeling that these networks contain Ure2p. In contrast, overexpressing wild-type cells show a variety of Ure2p distributions: usually, the protein is dispersed sparsely throughout the cytoplasm, although occasionally it is found in multiple small, focal aggregates. However, these distributions do not resemble the single, large networks seen in [URE3] cells, nor do the control cells exhibit cytoplasmic filaments. In [URE3] cell extracts, Ure2p is present in aggregates that are only partially solubilized by boiling in SDS and urea. In these aggregates, the NH2-terminal prion domain is inaccessible to antibodies, whereas the COOH-terminal nitrogen regulation domain is accessible. This finding is consistent with the proposal that the prion domains stack to form the filament backbone, which is surrounded by the COOH-terminal domains. These observations support and further specify the concept of the [URE3] prion as a self-propagating amyloid.

scholarly journals The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

1996 ◽  
Vol 16 (11) ◽  
pp. 6020-6028 ◽  
Author(s):  
S van Nocker ◽  
S Sadis ◽  
D M Rubin ◽  
M Glickman ◽  
H Fu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Protein ◽  
Degradation Pathway ◽  
26S Proteasome ◽  
Wild Type ◽  
Cell Extracts ◽  
Steady State Levels ◽  
Type Rate ◽  
Beta Galactosidase

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system

1985 ◽  
Vol 5 (9) ◽  
pp. 2361-2368
Author(s):  
L S Symington ◽  
P Morrison ◽  
R Kolodner
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Electrophoretic Mobility ◽  
Electrophoretic Analysis ◽  
Individual Band ◽  
Agarose Gels ◽  
Cell Extracts ◽  
Recombination System ◽  
Plasmid Recombination

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.

scholarly journals Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system.

1985 ◽  
Vol 5 (9) ◽  
pp. 2361-2368 ◽  
Author(s):  
L S Symington ◽  
P Morrison ◽  
R Kolodner
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Electrophoretic Mobility ◽  
Electrophoretic Analysis ◽  
Individual Band ◽  
Agarose Gels ◽  
Cell Extracts ◽  
Recombination System ◽  
Plasmid Recombination

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.

scholarly journals Ustilago Rabenhorstiana—An Alternative Natural Itaconic Acid Producer

Fermentation ◽  
2020 ◽  
Vol 6 (1) ◽  
pp. 4 ◽  
Author(s):  
Susan Krull ◽  
Malin Lünsmann ◽  
Ulf Prüße ◽  
Anja Kuenz
Keyword(s):  
Itaconic Acid ◽  
Aspergillus Terreus ◽  
Degradation Products ◽  
Basidiomycetous Yeasts ◽  
Fed Batch ◽  
Batch Mode ◽  
Renewable Feedstocks ◽  
The Family ◽  
Sugar Degradation ◽  
Shake Flasks

Itaconic acid is an industrial produced chemical by the sensitive filamentous fungus Aspergillus terreus and can replace petrochemical-based monomers for polymer industry. To produce itaconic acid with alternative renewable substrates, such as lignocellulosic based hydrolysates, a robust microorganism is needed due to varying compositions and impurities. Itaconic acid producing basidiomycetous yeasts of the family Ustilaginaceae provide this required characteristic and the species Ustilago rabenhorstiana was examined in this study. By an optimization of media components, process parameters, and a fed-batch mode with glucose the final titer increased from maximum 33.3 g·L−1 in shake flasks to 50.3 g·L−1 in a bioreactor. Moreover, itaconic acid was produced from different sugar monomers based on renewable feedstocks by U. rabenhorstiana and the robustness against weak acids as sugar degradation products was confirmed. Based on these findings, U. rabenhorstiana has a high potential as alternative natural itaconic acid producer besides the well-known U. maydis and A. terreus.

scholarly journals Identification of Kluyveromyces lactisTelomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain

1998 ◽  
Vol 18 (9) ◽  
pp. 4961-4970 ◽  
Author(s):  
Tracy Boswell Fulton ◽  
Elizabeth H. Blackburn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Telomerase Activity ◽  
Reaction Products ◽  
Telomeric Dna ◽  
Whole Cell ◽  
Cell Extracts ◽  
Dna Template ◽  
Insight Into

ABSTRACT Telomeres in the budding yeast Kluyveromyces lactisconsist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactistelomerase RNA, encoded by the TER1 gene, is ∼1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactiswhole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactistelomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactistelomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactistelomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.

Aryl-Substituted and Benzo-Annulated CycloSal-Derivatives of 2′,3′-Dideoxy-2′,3′-Didehydrothymidine Monophosphate — Correlation of Structure, Hydrolysis Properties and Anti-HIV Activity

2002 ◽  
Vol 13 (2) ◽  
pp. 129-141 ◽  
Author(s):  
Christian Ducho ◽  
Jan Balzarini ◽  
Lieve Naesens ◽  
Erik De Clercq ◽  
Chris Meier
Keyword(s):  
Antiviral Activity ◽  
Degradation Products ◽  
Hydrolytic Stability ◽  
Substitution Pattern ◽  
Cell Extracts ◽  
Hydrolysis Process ◽  
The Stability ◽  
Anti Hiv

The synthesis of phenyl-substituted and benzo-annulated cycloSal phosphate triesters of the nucleoside analogue 2′,3′-dideoxy-2′,3′-didehydrothymidine (d4T, Zerit™) as lipophilic, membrane-soluble pronucleotides is described. The cycloSal moiety was introduced by using cyclic chlorophosphite agents prepared from phenyl-substituted saligenin derivatives and orthohydroxymethylated naphthols, respectively. Hydrolysis studies (HPLC analysis) of the triesters 2, 3 showed a range of hydrolytic stability from 1.4 h up to 5.1 h and the stability could be correlated with the substitution pattern in the cycloSal moiety. A slight decrease of their stability was observed, if phenyl-substituted derivatives were hydrolyzed in human CEM/O cell extracts. D4T and thymine, possible products of enzymatic cleavage of the pronucleotides, were not detected in the cell extracts. A further investigation of the hydrolysis process was performed by 31P-NMR spectroscopy. This technique allowed a precise monitoring of the degradation products and the exact determination of the product ratio. Finally, the newly synthesized compounds were tested concerning their antiviral activity against HIV in vitro. A strong correlation of the hydrolysis properties and the antiviral activity was found. 3-phenyl- cycloSal-d4TMP showed a threefold increase in its anti-HIV-1 activity and retained full activity in thymidine kinase (TK) deficient cells, indicative of a successful TK-bypass.

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo

1988 ◽  
Vol 8 (1) ◽  
pp. 361-370
Author(s):  
S Ganguly ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Wild Type ◽  
Cell Extracts ◽  
Amber Suppressor ◽  
Gene Transcripts ◽  
A Site ◽  
Made In

We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene. Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu). Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced. The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro. Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts. A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site.

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