Über Zwischenprodukte der Riboflavin-Biosynthese bei Saccharomyces cerevisiae

1967 ◽  
Vol 22 (7) ◽  
pp. 755-758 ◽  
Author(s):  
F. Lingens ◽  
O. Oltmanns ◽  
A. Bacher
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Culture Fluid ◽  
Riboflavin Biosynthesis ◽  
Free Medium ◽  
Riboflavin Deficient

Using various riboflavin deficient mutants of S. cerevisiae, we have found two intermediates of the riboflavin biosynthesis. 6.7-Dimethyl-8-ribityl-lumazine was isolated from the culture fluid of two mutants. The formation of 4-ribitylamino-5-amino-uracil by two other mutants was proved by trapping the product with glyoxal or diacetyl, leading to the formation of 8-ribityl-lumazine or 6,7-dimethyl-8-ribityl-lumazine and riboflavin respectively. These mutants were able to grow in riboflavin free medium supplemented with diacetyl.6,7-Dimethyl-8-ribityl-lumazine promoted the growth of adequately blocked mutants.

scholarly journals Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Anna K. Wronska ◽  
Meinske P. Haak ◽  
Ellen Geraats ◽  
Eva Bruins Slot ◽  
Marcel van den Broek ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
De Novo ◽  
Laboratory Strain ◽  
Optimal Growth ◽  
Industrial Applications ◽  
Fast Growth ◽  
Growth Media ◽  
Free Medium ◽  
Content Type

ABSTRACT Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiae BIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae. This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiae. IMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae. Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.

scholarly journals Guanidine Hydrochloride Inhibits the Generation of Prion “Seeds” but Not Prion Protein Aggregation in Yeast

2002 ◽  
Vol 22 (15) ◽  
pp. 5593-5605 ◽  
Author(s):  
Frédérique Ness ◽  
Paulo Ferreira ◽  
Brian S. Cox ◽  
Mick F. Tuite
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Chaperone ◽  
Guanidine Hydrochloride ◽  
Free Medium ◽  
Seed Number ◽  
Cycle Model ◽  
Yeast Prions ◽  
Independent Process ◽  
Kinetics Of ◽  
Number Of Seeds

ABSTRACT [PSI +] strains of the yeast Saccharomyces cerevisiae replicate and transmit the prion form of the Sup35p protein but can be permanently cured of this property when grown in millimolar concentrations of guanidine hydrochloride (GdnHCl). GdnHCl treatment leads to the inhibition of the replication of the [PSI +] seeds necessary for continued [PSI +] propagation. Here we demonstrate that the rate of incorporation of newly synthesized Sup35p into the high-molecular-weight aggregates, diagnostic of [PSI +] strains, is proportional to the number of seeds in the cell, with seed number declining (and the levels of soluble Sup35p increasing) in the presence of GdnHCl. GdnHCl does not cause breakdown of preexisting Sup35p aggregates in [PSI +] cells. Transfer of GdnHCl-treated cells to GdnHCl-free medium reverses GdnHCl inhibition of [PSI +] seed replication and allows new prion seeds to be generated exponentially in the absence of ongoing protein synthesis. Following such release the [PSI +] seed numbers double every 20 to 22 min. Recent evidence (P. C. Ferreira, F. Ness, S. R. Edwards, B. S. Cox, and M. F. Tuite, Mol. Microbiol. 40:1357-1369, 2001; G. Jung and D. C. Masison, Curr. Microbiol. 43:7-10, 2001), together with data presented here, suggests that curing yeast prions by GdnHCl is a consequence of GdnHCl inhibition of the activity of molecular chaperone Hsp104, which in turn is essential for [PSI +] propagation. The kinetics of elimination of [PSI +] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for “prionization” of Sup35p in [PSI +] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates.

scholarly journals A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism.

1997 ◽  
Vol 8 (7) ◽  
pp. 1273-1291 ◽  
Author(s):  
P Chen ◽  
J D Choi ◽  
R Wang ◽  
R J Cotter ◽  
S Michaelis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Culture Fluid ◽  
Proteolytic Processing ◽  
Atp Binding ◽  
Related Peptide ◽  
Atp Binding Cassette Transporter ◽  
Independent Mechanism ◽  
Cellular Machinery ◽  
Atp Binding Cassette

Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms. The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself. We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl- and carboxylmethylcysteine modifications. The formation and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl1) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor. Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-dependent but length-specific proteolytic activity. Third, a-factor and AFRP use distinct cellular machinery for their secretion. Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion. Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor. Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.

scholarly journals Inositol transport in Saccharomyces cerevisiae is regulated by transcriptional and degradative endocytic mechanisms during the growth cycle that are distinct from inositol-induced regulation.

1996 ◽  
Vol 7 (1) ◽  
pp. 81-89 ◽  
Author(s):  
K S Robinson ◽  
K Lai ◽  
T A Cannon ◽  
P McGraw
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Growth Cycle ◽  
Endocytic Pathway ◽  
Negative Regulator ◽  
Lag Phase ◽  
Free Medium ◽  
Uptake Activity ◽  
Inositol Uptake

Regulation of inositol uptake activity in Saccharomyces cerevisiae during the growth cycle was examined. Activity increased as the cell population transited from lag phase to exponential growth, and continued to increase until late exponential phase. The increase in activity was due to increased transcription of the ITR1 gene and synthesis of the Itr1 permease. When the culture reached stationary phase, uptake activity decreased and dropped to a minimum within 4 h. The decrease was due to repression of ITR1 transcription, independent of the negative regulator Opi1p, and degradation of the existing permease. Degradation depended on delivery of the permease to the vacuole through the END3/END4 endocytic pathway. During exponential growth in inositol-containing medium the permease is also rapidly degraded, whereas in inositol-free medium the permease is highly stable. Rapid degradation of the permease at stationary phase occurred in inositol-free medium, indicating that there are two distinct mechanisms that trigger endocytosis and degradation in response to different physiological stimuli. In addition, the level of the enzyme required for inositol biosynthesis, inositol-1-phosphate synthase, encoded by INO1, is not reduced in stationary-phase cells, and this contrast in the regulation of inositol supply is discussed.

scholarly journals Nutrient regulation of oligopeptide transport in Saccharomyces cerevisiae

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 3133-3145 ◽  
Author(s):  
Amy M. Wiles ◽  
Houjian Cai ◽  
Fred Naider ◽  
Jeffrey M. Becker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Environmental Conditions ◽  
Transport Systems ◽  
Free Medium ◽  
Small Peptides ◽  
Functional Protein ◽  
Nutrient Regulation ◽  
Free Environment

Small peptides (2–5 amino acid residues) are transported into Saccharomyces cerevisiae via two transport systems: PTR (Peptide TRansport) for di-/tripeptides and OPT (OligoPeptide Transport) for oligopeptides of 4–5 amino acids in length. Although regulation of the PTR system has been studied in some detail, neither the regulation of the OPT family nor the environmental conditions under which family members are normally expressed have been well studied in S. cerevisiae. Using a lacZ reporter gene construct fused to 1 kb DNA from upstream of the genes OPT1 and OPT2, which encode the two S. cerevisiae oligopeptide transporters, the relative expression levels of these genes were measured in a variety of environmental conditions. Uptake assays were also conducted to measure functional protein levels at the plasma membrane. It was found that OPT1 was up-regulated in sulfur-free medium, and that Ptr3p and Ssy1p, proteins involved in regulating the di-/tripeptide transporter encoding gene PTR2 via amino acid sensing, were required for OPT1 expression in a sulfur-free environment. In contrast, as measured by response to toxic tetrapeptide and by real-time PCR, OPT1 was not regulated through Cup9p, which is a repressor for PTR2 expression, although Cup9p did repress OPT2 expression. In addition, all of the 20 naturally occurring amino acids, except the sulfur-containing amino acids methionine and cysteine, up-regulated OPT1, with the greatest change in expression observed when cells were grown in sulfur-free medium. These data demonstrate that regulation of the OPT system has both similarities and differences to regulation of the PTR system, allowing the yeast cell to adapt its utilization of small peptides to various environmental conditions.

Isolierung von Riboflavin-Mangelmutanten von Saccharomyces cerevisiae

1967 ◽  
Vol 22 (7) ◽  
pp. 751-754 ◽  
Author(s):  
O. Oltmanns ◽  
F. Lingens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Dependence ◽  
Yeast Extract ◽  
Yeast Extract Peptone Dextrose ◽  
Riboflavin Deficient

Riboflavin-deficient mutants of S. cerevisiae, not known hitherto, were isolated using a yeast extract-peptone-dextrose medium supplemented with 10 γ riboflavin/ml. After treatment with ethylmethanesulfonate, 0,4% of the surviving cells or 2% of the isolated biochemical mutants were deficient of riboflavin.Seven of 14 riboflavin deficient mutants were tested for temperature dependence but no such dependence was found. Two mutants accumulated a green fluorescing substance. Cross-feeding tests were not successful.

Exogeneous factors are not necessary in attachment, spreading and polarization of hela cells

1978 ◽  
Vol 36 (2) ◽  
pp. 310-311
Author(s):  
W. Liebrich
Keyword(s):  
Hela Cells ◽  
Calf Serum ◽  
Glass Tube ◽  
Serum Free Medium ◽  
Nacl Solution ◽  
Free Medium ◽  
Minimum Essential Medium ◽  
Serum Free ◽  
All Solutions ◽  
Glass Support

HeLa cells were grown for 2-3 days in EAGLE'S minimum essential medium with 10% calf serum (S-MEM; Seromed, München) and then incubated for 24 hours in serum free medium (MEM). After detaching the cells with a solution of 0. 14 % EDTA and 0. 07 % trypsin (Difco, 1 : 250) they were suspended in various solutions (S-MEM = control, MEM, buffered salt solutions with or without Me++ions, 0. 9 % NaCl solution) and allowed to settle on glass tube slips (Leighton-tubes). After 5, 10, 15, 20, 25, 30, 1 45, 60 minutes 2, 3, 4, 5 hours cells were prepared for scanning electron microscopy as described by Paweletz and Schroeter. The preparations were examined in a Jeol SEM (JSM-U3) at 25 KV without tilting.The suspended spherical HeLa cells are able to adhere to the glass support in all solutions. The rate of attachment, however, is faster in solutions without serum than in the control. The latter is in agreement with the findings of other authors.

Immunogold labeling of Pneumocystis carinii for Electron microscopy

1991 ◽  
Vol 49 ◽  
pp. 270-271
Author(s):  
Michael P. Goheen ◽  
Marilyn S. Bartlett ◽  
James W. Smith
Keyword(s):  
Electron Microscopy ◽  
Pneumocystis Carinii ◽  
Severe Pneumonia ◽  
Culture Fluid ◽  
Immunogold Labeling ◽  
Antigenic Sites ◽  
Transmission Electron ◽  
Response To Chemotherapy ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome

Studies of the biology of Pneumocystis carinii (PC) are of increasing importance because this extracellular pathogen is a frequent source of severe pneumonia in patients with acquired immunodeficiency syndrome (AIDS) and is a leading cause of mortality in these patients. Immunoelectron microscopic localization of antigenic sites on the surface of PC would improve the understanding of these sites and their role in pathenogenisis of the disease and response to chemotherapy. The purpose of this study was to develop a methodology for visualizing immunoreactive sites on PC with transmission electron microscopy (TEM) using immunogold labeled probes.Trophozoites of PC were added to spinner flask cultures and allowed to grow for 7 days, then aliquots of tissue culture fluid were centrifuged at 12,000 RPM for 30 sec. Pellets of organisims were fixed in either 1% glutaraldehyde, 0.1% glutaraldehyde-4% paraformaldehyde, or 4% paraformaldehyde for 4h. All fixatives were buffered with 0.1M Na cacodylate and the pH adjusted to 7.1. After fixation the pellets were rinsed in 0.1M Na cacodylate (3X), dehydrated with ethanol, and immersed in a 1:1 mixture of 95% ethanol and LR White resin.

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