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1
Swiegers, Jan Hendrik. "Carnitine in yeast and filamentous fungi." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/49753.
Testo completoAbstract (sommario):
Dissertation (PhD)--University of Stellenbosch, 2003.<br>ENGLISH ABSTRACT: In the yeast Saccharomyces cerevtstee, two biochemical pathways ensure that
activated cytoplasmic or peroxisomal acetyl-groups are made available for
mitochondrial energy production when the cells utilise non-fermentable carbon
sources. The first pathway is the glyoxylate cycle, where two activated acetyl-groups
are incorporated into each cycle, which releases a C4 intermediate. This intermediate
is then transported to the mitochondria where it can enter the tricarboxylic acid cycle.
The second pathway is the carnitine shuttle. Activated acetyl-groups react with
carnitine to form acetylcarnitine, which is then transported to the mitochondria where
the acetyl group is transferred.
In this study it was shown that the deletion of the glyoxylate cycle specific citrate
synthase, encoded by CIT2, results in a strain that is dependent on carnitine for
growth on non-fermentable carbon sources. Using a /::"cit2 strain, mutants affected in
carnitine-dependent metabolic activities were generated. Complementation of the
mutants with a genomic library resulted in the identification of four genes involved in
the carnitine shuttle. These include: (i) the mitochondrial and peroxisomal carnitine
acetyltransferase, encoded by CAT2; (ii) the outer-mitochondrial carnitine
acetyltransferase, encoded by YA T1; (iii) the mitochondrial carnitine translocase,
encoded by CRC1; and (iv) a newly identified carnitine acetyltransferase, encoded by
YAT2. All three carnitine acetyltransferases are essential in a carnitine-dependent
strain.
The dependence on exogenous carnitine of the /::"cit2 strain when grown on nonfermentable
carbon sources suggested that S. cerevisiae does not biosynthesise
carnitine. Measurements using electrospray mass spectrometry confirmed this
hypothesis. As a result an investigation was initiated into carnitine biosynthesis in
order to genetically engineer a S. cerevisiae strain that could endogenously
biosynthesise carnitine.
The filamentous fungus, Neurospora crassa, was one of the first organisms used
in the seventies to identify the precursor and intermediates of carnitine biosynthesis.
However, it was only about twenty years later that the first genes encoding these
enzymes where characterised. Carnitine biosynthesis is a four-step process, which
starts with trimethyllysine as precursor. Trimethyllysine is converted to hydroxytrimethyllysine
by the enzyme trimethyllysine hydroxylase (TMLH). Hydroxytrimethyllysine
is cleaved to trimethylamino-butyraldehyde by the
hydroxytrimethyllysine aldolase (HTMLA) releasing glycine. Trimethylaminobutyraldehyde
is dehydrogenated to trimethylamino-butyrate (y-butyrobetaine) by
trimethylamino-butyraldehyde dehydrogenase (TMABA-DH). In the last step, ybutyrobetaine
is converted to t-carnltine by y-butyrobetaine hydroxylase (BBH).
The N. crassa TMLH homologue was identified in the genome database based
on the protein sequence homology of the human TMLH. Due to the high amount of introns predicted for this gene, the cDNA was cloned and subjected to sequencing,
which then revealed that the gene indeed had seven introns. Functional expression
of the gene in S. cerevisiae and subsequent enzymatic analysis revealed that the
gene coded for a TMLH. It was therefore named cbs-1 for "carnitine biosynthesis
gene no. 1JJ. Most of the kinetic parameters were similar to that of the human TMLH
enzyme. Following this, a genomic copy of the N. crassa BBH homologue was cloned
and functionally expressed in S. cerevisiae. Biochemical analysis revealed that the
BBH enzyme could biosynthesise L-carnitine from y-butyrobetaine and the gene was
named cbs-2. In addition, the gene could rescue the growth defect of the carnitinedependent
Scii? strain on non-fermentable carbon sources when y-butyrobetaine was
present. This is the first report of an endogenously carnitine biosynthesising strain of
S. cerevisiae.
The cloning of the remaining two biosynthesis genes presents particular
challenges. To date, the HTMLA has not been characterised on the molecular level
making the homology-based identification of this protein in N. crassa impossible.
Although the TMABA-DH has been characterised molecularly, the protein sequence
is conserved for its function as a dehydrogenase and not conserved for its function in
carnitine biosynthesis, as in the case of TMLH and BBH. The reason for this is
probably due to the fact that the enzyme is involved in other metabolic processes.
The use of N. crassa carnitine biosynthesis mutants would probably be one way in
which to overcome these obstacles.
The !1cit2 mutant proved useful in studying carnitine related metabolism. We
therefore searched for suppressors of !1cit2, which resulted in the cloning of RAS2. In
S. cerevisiae, two genes encode Ras proteins, RAS1 and RAS2. GTP-bound Ras
proteins activate adenylate cyclase, Cyr1 p, which results in elevated cAMP levels.
The cAMP molecules bind to the regulatory subunit of the cAMP-dependent kinase
(PKA), Bcy1 p, thereby releasing the catalytic subunits Tpk1 p, Tpk2p and Tpk3p. The
catalytic subunits phosphorylate a variety of regulators and enzymes involved in
metabolism. Overexpression of RAS2 could suppress the growth defect of the Sclt?
mutant on glycerol. In general, overexpression of RAS2 enhanced the proliferation of
wild-type cells grown on glycerol. However, the enhancement of proliferation was
much better for the !1cit2 strain grown on glycerol. In this respect, the retrograde
response may play a role. Overexpression of RAS2 resulted in elevated levels of
intracellular citrate and citrate synthase activity. It therefore appears that the
suppression of !1cit2 by RAS2 overexpression is a result of the general upregulation
of the respiratory capacity and possible leakage of citrate and/or citrate synthase
from the mitochondria. The phenotype of RAS2 overexpression contrasts with the
hyperactive RAS2val19 allele, which causes a growth defect on glycerol. However,
both RAS2 overexpression and RAS2val19activate the cAMP/PKA pathway, but the
RAS2val19dependent activation is more severe. Finally, this study implicated the
Ras/cAMP/PKA pathway in the proliferation effect on glycerol by showing that in a
Mpk1 strain, the growth effect is blocked. However, the enhanced proliferation was still observed in the Mpk2 and Mpk3 strains when RAS2 was overexpressed.
Therefore, it seems that Tpk1 p plays an important role in growth on non-fermentable
carbon sources, a notion that is supported by the literature.<br>AFRIKAANSE OPSOMMING: In die gis Saccharomyces cerevtstee, is daar twee metaboliese weë waarmee
geaktiveerde asetielgroepe na die mitochondrium vervoer kan word wanneer die sel
op nie-fermenteerbare koolstofbronne groei. Die een weg is die glioksilaatsiklus,
waar die geaktiveerde asetielgroepe geïnkorporeer word in die siklus en dan
vrygestel word as Ca-intermediêre. Hierdie intermediêre word dan na die
mitochondrium vervoer waar dit in die trikarboksielsuursiklus geïnkorporeer word. Die
ander weg is die karnitiensiklus, waar geaktiveerde asetielgroepe met karnitien
reageer om asetielkarnitien te vorm wat dan na die mitochondrium vervoer word waar
dit die asetielgroep weer vrygestel.
Hierdie studie het getoon dat die delesie van die glioksilaatsiklus spesifieke
sitraatsintetase, gekodeer deur CIT2, die gisras afhanklik maak van karnitien vir groei
op nie-fermenteerbare koolstofbronne. Deur gebruik te maak van 'n ócit2 gisras, kon
mutante, wat geaffekteer is in karnitien-verwante metaboliese aktiwiteite, gegenereer
word. Komplementering van die mutante met 'n genomiese biblioteek het gelei tot die
identifisering van vier gene betrokke by die karnitiensiklus. Hierdie gene sluit in: (i)
die mitochondriale en die peroksisomale karnitienasetieltransferase, gekodeer deur
CAT2; (ii) die buite-mitochondriale karnitienasetieltransferase, gekodeer deur YAT1;
(iii) die mitochondriale karnitientranslokase, gekodeer deur CRC1; en (iv) 'n nuutgeïdentifiseerde
karnitienasetieltransferase, gekodeer deur YAT2. Daar benewens, is
ook gewys dat al drie karnitienasetieltransferases noodsaaklik is in 'n karriltienafhanklike
gisras.
Die afhanklikheid van eksogene karnitien van die ócit2 gisras, wanneer dit
gegroei word op nie-fermenteerbare koolstofbronne, was aanduidend dat
S. cerevisiae nie karnitien kan biosintetiseer nie. Metings deur middel van
elektronsproeimassaspektrometrie het hierdie veronderstelling bevestig. Gevolglik is
'n ondersoek deur ons geïnisieer in die veld van karnitienbiosintese om 'n
S. cerevisiae gisras geneties te manipuleer om karnitien sodoende endogenies te
biosintetiseer.
Die filamentagtige fungus, Neurospora crassa, was een van die eerste
organismes wat in die sewentiger jare gebruik is om die voorloper en intermediêre
van karnitienbiosintese te identifiseer. Dit was egter eers sowat twintig jaar later dat
die eerste gene wat vir hierdie ensieme kodeer, gekarakteriseer is.
Karnitienbiosintese is 'n vierstap-proses wat met trirnetlellisten as voorloper begin.
Trimetiellisien word omgeskakel na hidroksi-trimetiellisien deur die ensiem
trimetiellisienhidroksilase (TMLH). Hidroksietrimetlelllsien word dan gesplits om
trimetielaminobuteraldehied te vorm deur die werking van die
hidroksitrimetiellisienaldolase (HTMLA) met die gevolglike vrystelling van glisien.
Trimetielaminobuteraldehied word dan na trimetielaminobuteraat (y-butirobeteïen)
deur trimetielaminobuteraldehied dehidrogenase (TMABA-DH) gedehidrogeneer. In die laaste stap word y-butirobeteïen deur middel van die y-butirobeteïen hidroksilase
(BBH) na L-karnitien omgeskakel.
Op grond van die proteïenvolgordehomologie in die genoomdatabasis tussen die
menslike TMLH en N. crassa se TMLH is laasgenoemde geïdentifiseer. As gevolg
van die groot getal introns wat vir hierdie geen voorspel is, is die cDNA-weergawe
daarvan gekloneer en aan volgordebepaling onderwerp. Dit het getoon dat die geen
inderdaad sewe introns bevat. Funksionele uitdrukking van die geen in S. cerevisiae
en ensiematiese analise het getoon dat die geen vir 'n TMLH kodeer en is gevolglik
cbs-1 genoem; dit staan vir "karnitien biosintese geen no. 1tt. Meeste van die
kinetiese parameters was ook soortgelyk aan die van die menslike TMLH-ensiem.
Hierna is 'n genomiese kopie van N. crassa se BBH-homoloog gekloneer en
funksioneel in S. cerevisiae uitgedruk. Biochemiese analise het getoon dat die
uitgedrukte BBH-ensiem L-karnitien vanaf y-butirobeteïen kan biosintetiseer en die
geen is cbs-2 genoem. Daar benewens kon die geen die groeidefek van die
karnitien-afhanklike tlcit2-gisras ophef wanneer dit op nie-fermenteerbare
koolstofbronne in die teenwoordigheid van y-butirobeteïen aangekweek is. Hierdie is
die eerste verslag oor 'n endogeniese karnitien-biosintetiserende ras van
S. cerevisiae.
Die klonering van die oorblywende twee karnitienbiosintetiserende gene het
sekere uitdagings. Tot op datum, is die HTMLA nog nie tot op genetiese vlak
gekarakteriseer nie, wat dan die homologie-gebaseerde identifikasie van hierdie
proteïen in N. crassa onmoontlik maak. Alhoewel die TMABA-DH geneties
gekarakteriseer is, is die proteïenvolgorde ten opsigte van sy funksie as 'n
dehidrogenase gekonserveer, maar nie vir sy funksie in karnitienbiosintese soos in
die geval van TMLH en BBH nie. Die rede hiervoor is moontlik omdat die ensiem ook
in ander metaboliese prosesse betrokke is. Die gebruik van N. crassa
karnitienmutante sal moontlik een manier wees om hierdie probleme te oorkom.
Die tlcit2-mutant het handig te pas gekom vir die bestudering van karnitienverwante
metabolisme. Dus is daar vir onderdrukkers van die tlcit2-mutant gesoek
wat gelei het tot die klonering van die RAS2-geen. In S. cere visiae , kodeer twee
gene vir Ras-proteïene, RAS1 en RAS2. GTP-gebonde Ras-proteïene aktiveer
adenilaatsiklase, Cyr1 p, wat verhoogde intrasellulêre cAMP-vlakke tot gevolg het.
Die cAMP bind aan die regulatoriese subeenheid van die cAMP-proteïenkinase
(PKA), Bcy1 p, en daardeur word die katalitiese subeenhede, Tpk1 p, Tpk2p en
Tpk3p, vrygestel. Die katalitiese subeenheid fosforileer 'n verskeidenheid van
reguleerders en ensieme betrokke by metabolisme. Ooruitdrukking van RAS2 het die
groeidefek van die tlcit2-mutant op gliserolonderdruk. Oor die algemeen, verbeter
die ooruitdrukking van RAS2 die proliferasie van die wildetipe op gliserol bevattende
media. Alhoewel, die verbetering van proliferasie was baie meer opmerklik in die
tlcit2-gisras. In hierdie verband, speel die gedegenereerde response dalk 'n rol.
Ooruitdrukking van RAS2 het verhoogde intrasellulêre vlakke van sitraat- en
sitraatsintetase-aktiwiteit tot gevolg gehad. Dit wou dus voorkom asof die onderdrukking van die ócit2-groeidefek deur RAS2 se ooruitdrukking die gevolg was
van algemene opreguiering van respiratoriese kapasiteit en die lekkasie van sitraat
en/of sitraatsintetase uit die mitochondria. Die fenotipe van RAS2 ooruitdrukking
kontrasteer die hiperaktiewe RAS2va
/
19 alleel, wat 'n groeidefek op gliserol media
veroorsaak. Alhoewel beide RAS2-00ruitdrukking en RAS2va
/
19 die cAMP/PKA-weg
aktiveer, is gevind dat die RAS2va/19-afhanklike aktivering strenger is. Ten slotte, die
cAMP/PKA-weg is in die proliferasie effek op gliserol media geïmpliseer deur te wys
dat in 'n Mpk1-gisras, die groeieffek geblokkeer is. Alhoewel, die verbeterde
proliferasie is steeds waargeneem in die Mpk2-en Mpk3-gisrasse toe die RAS2-geen
ooruitgedruk is. Dus, dit wil voorkom asof Tpk1 p 'n belangrike rol in die groei van
gisselle op nie-fermenteerbare koolstofbronne speel; 'n veronderstelling wat deur die
literatuur ondersteun word.
2
Cao, Juxiang Locy Robert D. "Functional genomics of GABA metabolism in yeast thermotolerance." Auburn, Ala, 2008. http://repo.lib.auburn.edu/2007%20Fall%20Dissertations/Cao_Juxiang_41.pdf.
Testo completo
3
Rome, Jacqueline Louise de. "Biosorption of heavy metals by fungi and yeast." Thesis, University of Dundee, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338281.
Testo completo
4
Brady, Dean. "Bioaccumulation of metal cations by yeast and yeast cell components." Thesis, Rhodes University, 1993. http://hdl.handle.net/10962/d1004107.
Testo completoAbstract (sommario):
The aim of the project was to determine whether a by-product of industrial fermentations, Saccharomyces cerevisiae, could be utilized to bioaccumulate heavy metal cations and to partially define the mechanism of accumulation. S. cerevisiae cells were found to be capable of accumulating Cu²⁺in a manner that was proportional to the external Cu²⁺ concentration and inversely proportional to the concentration of biomass. The accumulation process was only minimally affected by temperature variations between 5 and 40°C or high ambient concentrations of sodium chloride. The accumulation process was however considerably affected by variations in pH, bioaccumulation being most efficient at pH 5 - 9 but becoming rapidly less so at either extreme of pH. Selection for copper resistant or tolerant yeast diminished the yeast's capacity for Cu²⁺ accumulation. For this and other reasons the development of heavy metal tolerance in yeasts was deemed to be generally counterproductive to heavy metal bioaccumulation. The yeast biomass was also capable of accumulating other heavy metal cations such as c0²⁺ or Cd²⁺. The yeast biomass could be harvested after bioaccumulation by tangential filtration methods, or alternatively could be packed into hollow fibre microfilter membrane cartridges and used as a fixed-bed bioaccumulator. By immobilizing the yeast in polyacrylamide gel and packing this material into columns, cu²⁺, C0²⁺ or Cd²⁺ could be removed from influent aqueous solutions yielding effluents with no detectable heavy metal, until breakthrough point was reached. This capacity was hypothesized to be a function of numerous "theoretical plates of equilibrium" within the column. The immobilized biomass could be eluted with EDTA and recycled for further bioaccumulation processes with minor loss of bioaccumulation capacity. Yeast cells were fractionated to permit identification of the major cell fractions and molecular components responsible for metal binding. Isolation of the yeast cell walls permitted investigation of their role in heavy metal accumulation. Although the amino groups of chitosan and proteins, the carboxyl groups of proteins, and the phosphate groups of phosphomannans were found to be efficient groups for the accumulation of copper, the less effective hydroxyl groups of the carbohydrate polymers (glucans and mannans) had a similar overall capacity for copper accumulation owing to their predominance in the yeast cell wall. The outer (protein-mannan) layer of the yeast cell wall was found to be a better Cu²⁺ chelator than the inner (chitinglucan) layer. It appeared that the physical condition of the cell wall may be more important than the individual macromolecular components of the cell wall in metal accumulation. It was apparent that the cell wall was the major, if not the sole contributor to heavy metal accumulation at low ambient heavy metal concentrations. At higher ambient metal concentrations the cytosol and vacuole become involved in bioaccumulation. Copper and other metals caused rapid loss of 70% of the intracellular potassium, implying permeation of the plasma membrane. This was followed by a slower "leakage" of magnesium from the vacuole which paralleled Cu²⁺ accumulation, suggesting that it may represent some form of ion-exchange. An intracellular copper chelating agent of approximately 2 kDalton molecular mass was isolated from copper tolerant yeast. This chelator was not a metallothionein and bound relatively low molar equivalents of copper compared to those reported for metallothionein. Treatment of the biomass with hot alkali yielded two biosorbents, one soluble (which could be used as a heavy metal flocculent), and an insoluble biosorbent which could be formed into a granular product to be used in fixed-bed biosorption columns. The granular biosorbent could accumulate a wide range of heavy metal cations in a semispecific manner and could be stored in a dehydrated form indefinitely, and rehydrated when required. Bioaccumulation by live algae was investigated as an alternative to yeast based processes. Various strains of algae, of which Scenedesmus and Selenastrum were the most effective, were found to be capable of accumulating heavy metals such as Cu²⁺, Pb²⁺ and Cr³⁺.
5
Beh, Ai Lin Chemical Sciences & Engineering Faculty of Engineering UNSW. "Investigation of yeasts and yeast-like fungi associated with Australian wine grapes using cultural and molecular methods." Awarded by:University of New South Wales. Chemical Sciences & Engineering, 2007. http://handle.unsw.edu.au/1959.4/40683.
Testo completoAbstract (sommario):
This thesis presents a systematic investigation ofyeasts associated with wine grapes cultivated in several Australian vineyards during the 2001-2003 vintages. Using a combination of cultural and molecular methods, yeast populations of red (Cabernet sauvignon, Merlot, Tyrian) and white (Sauvignon blanc, Semilion) grape varieties were examined throughout grape cultivation. The yeast-like fungus, Aureobasidium pullulans, was the most prevalent species found on grapes. Various species of Cryptococcus, Rhodotorula and Sporobolomyces were frequently isolated throughout grape maturation. Ripe grapes showed an increased incidence of Hanseniaspora and Metschnikowia species for the 2001-2002 seasons, but not for the drought affected, 2002-2003 seasons. Atypical, hot and dry conditions may account for this difference in yeast flora and have limited comparisons of data to determine the influences of vineyard location, grape variety and pesticide applications on the yeast ecology. More systematic and controlled studies of these variables are required. Damaged grape berries harboured higher yeast populations and species diversity than intact healthy berries. PCR-DGGE analysis was less sensitive than plate culture for describing the diversity of yeast species on grapes; it detected prevalent species, but subdominant populations below 103 CFU/g were not detected. In some cases, PCR-DGGE revealed the presence ofyeasts (Candida galli, C. zemplinina) not isolated by culture. Fermentative wine species (Kluyveromyces, Torulaspora, Saccharomyces) were rarely isolated, and only detected by enrichment cultures. Significant morphological and genetic variability were detected among A. pullulans and other black yeasts isolates from grapes. Taxonomic characterization of 61 strains by ITS-RFLP and rDNA sequencing revealed that they belonged to several distinct species within the generic groupings ofAureobasidium, Hormonema and Kabatiella. Isolates were strong producers of extracellular enzymes and polysaccharides that could have oenological significance, and, using a plate assay, some were antagonistic towards Bacillus thuringiensis, several wine yeasts, and some spoilage and mycotoxigenic fungi found on grapes. Growth of Saccharomyces cerevisiae was not inhibited by these organisms in grape juice. A species-specific probe was developed for the identification of the wine spoilage yeast, Zygosaccharomyces bailii in a microtitre plate hybridization assay. The probe detected 102 cells/ml in wine, reliably differentiating Z. bailii from other Zygosaccharomyces and other wine-related yeasts.
6
Ha, Seon-Ah. "The role of the INP53 protein in membrane trafficking in yeast /." free to MU campus, to others for purchase, 2002. http://wwwlib.umi.com/cr/mo/fullcit?p3060102.
Testo completo
7
Yip, Hopi. "Genetic manipulation of baker's yeast for improved maltose utilisation /." [Richmond, N.S.W.] : Centre for Biostructural and Biomolecular Resarch, Faculty of Science and Technolocy, University of Western Sydney, Hawkesbury, 1999. http://library.uws.edu.au/adt-NUWS/public/adt-NUWS20030625.100807/index.html.
Testo completo
8
Soerensen, Tine Kring. "Cloning and characterisation of a gpt gene from Aspergillus niger." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364397.
Testo completo
9
Buchan, Arlene. "The roles of calcium and calmodulin in the regulation of dimorphism and pathogenicity of Candida albicans." Thesis, University of Aberdeen, 1995. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU083184.
Testo completoAbstract (sommario):
Studies on the capacity of growing and non-growing cells to be induced to grow in different morphological forms suggested that metabolically active cells are better able to undergo dimorphism than metabolically quiescent cells. Accelerated growth kinetics requiring de novo RNA and or protein biosynthesis but not DNA synthesis were observed in metabolically active cells undergoing a yeast to hyphal transition. The roles that divalent cation calcium plays in eukaryotic growth and morphogenesis are illustrated in this study with regard to C. albicans. Free calcium was required for germ tube emergence from stationary phase yeast cells but not for subsequent growth or hyphal extension. The galvanotropic response of C. albicans hyphae was investigated and was shown to be quantitatively dependent on the concentration of available calcium in the medium and also on the integrity of calcium dependent second messenger systems. The mechanism of galvanotropism of C. albicans may be due to the electrophoretic movement of charged proteins, possibly calcium transport channels, in the plasma membrane as shown by the dependence of cathodotropic growth on external pH. Although the yeast to hyphal transition can be easily controlled and studied in vitro, attempts to use such methods to control morphological development, with a view to comparing the relative infectivity of each morphology, are complicated in the environment in vivo. Yeast cells were introduced into the rat vagina, or systemically into mice, along with chemical effectors which had been shown to prevent the yeast to mycelial transition in vitro without affecting the growth rate of the cells per se. It was concluded tentatively from this study, that, in the rat vaginal model of candidosis, the hyphal form of growth is better adapted and more pathogenic than the yeast form.
10
Beckhouse, Anthony Gordon Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "The transcriptional and physiological alterations in brewers yeast when shifted from anaerobic to aerobic growth conditions." Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2006. http://handle.unsw.edu.au/1959.4/24201.
Testo completoAbstract (sommario):
Yeast are exposed to many physical and chemical stresses when used in large-scale industrial fermentations, particularly the initial stages in which yeast are shifted from anaerobic storage to aerated wort. This work investigated the transcriptional and physiological responses of yeast that had been shifted from anaerobic to aerobic growth conditions. Microarray technology was employed to determine the transcriptional changes that occurred in the first hour of a pilot-plant fermentation compared to the 23rd hour. It was found that over 100 genes were up-regulated initially including genes involved in the synthesis of the essential membrane sterol ergosterol and genes for the protection of cells against oxidative stress. It was also determined that cells which accumulate ergosterol precursors in the absence of ergosterol were more sensitive to exogenous oxidative stresses, indicating a role for ergosterol in oxidative stress tolerance. Aeration of anaerobically grown cells did not affect their growth kinetics or viability. However, anaerobically grown cells were hypersensitive to exogenous oxidative stress compared to their aerobic counterparts. Anaerobic cells that underwent a short period of aeration prior to treatment with hydrogen peroxide generated a tolerance to the oxidant, indicating that the period of aeration produced an adaptive-like response. Microarray analysis of the cells during the period of aeration showed that representative genes from the oxidative stress response family were up-regulated rapidly and it was determined that the response was controlled by the Yap1p and Skn7p transcription factors. Deletion of the transcription factor genes indicated that they were responsible for the creation of tolerance to oxidant. Target gene products of the two transcription factors (Gpx2p, Gsh1p and Trx2p) were shown to be induced during the shift to aeration; however, the glutathione redox balance did not seem to be affected as the cells were shifted from highly reduced to oxidising environments. Unexpectedly, it was discovered that genes involved in the synthesis of amino acids were up-regulated during anaerobic growth and stringently downregulated upon aeration of cells. The transcriptional activator of those genes (Gcn4p) was essential for growth in anaerobic media which included amino acid supplementation.
11
Mocke, Bernard A. "The breeding of yeast strains for novel oenological outcomes." Thesis, Link to the online version, 2005. http://hdl.handle.net/10019/1117.
Testo completo
12
Trollope, Kim. "Investigation of resveratrol production by genetically engineered Saccharomyces cervisiae strains /." Link to the online version, 2006. http://hdl.handle.net/10019/1247.
Testo completo
13
Speers, Robert Alexander. "Rheological and colloidal properties of commercial brewing yeast suspensions." Thesis, University of British Columbia, 1991. http://hdl.handle.net/2429/31517.
Testo completoAbstract (sommario):
A three part study was carried out to examine rheological, colloidal and floe microstructural aspects of industrial brewing yeast strains. Following a review of the literature, the rheological properties of four yeast strains (two flocculent ale and lager types and their non-flocculent variants) were examined. In related colloidal studies, orthokinetic flocculation rates of these strains as well as their cell surface charge were determined. Floc microstructure was characterized using both light and scanning electron microscopy. In a summary chapter, the cell floc model (a modification of Hunter's elastic floc model) was used to the explain the rheological and colloidal behaviour of brewing yeast suspensions.
Flow behaviour studies of the commercial yeast suspensions suspended in a calcium-containing sodium acetate buffer revealed that yeast flocculent characteristics had an important influence on their suspension flow behaviour. As cell concentrations increased, suspension flow properties become increasingly non-Newtonian and could be described by the Casson model at low rates of shear and the Bingham model at shear rates above 100 s⁻¹. The cell floc model was proposed to explain the Bingham flow behaviour of these csuspensions. The Bingham yield stress in these suspensions was believed to be a function of the orthokinetic capture coefficient, cell volume and the energy to break up doublet cells. Increasing temperature tended to lower the Bingham yield stress in lager strains and increase the yield stress in ale strains. A semi-empirical explanation for the viscosity increase of deflocculated cell suspensions and the estimation of pseudo-capture coefficients was presented.
Furthermore, studies of the flow behaviour of yeast strains suspended in decarbonated ale and lager beer revealed that: 1) suspensions of flocculent strains show
higher yield stress values than their non-flocculent variants, 2) ale strain suspensions tended to have higher yield values than the lager strains and 3) yeast dispersed in beer had higher yield stress values than when suspended in buffered calcium suspensions. This last observation was believed to reflect the influence of ethanol on the cell binding process which has important implications for future measurements of yeast flocculation.
Colloidal studies revealed for the first time, that the orthokinetic rate of flocculation of brewing yeast cells could be modelled by a first order equation, as predicted by fundamental colloid theory. While subject to considerable variation, measured rate constants led to the calculation of orthokinetic capture coefficients. Yeast cell zeta potential values generally agreed with literature data but could not be employed in the DLVO model of colloid flocculation to explain measured orthokinetic capture coefficient values. Examination of the cell zeta potential data indicated that the data had non-normal distributions.
SEM examination of the four industrial yeast strains suggested that a number of distinct structures mediated cell-to-cell interaction and that intra-strain differences occurred. These findings, along with the observation of non-normal surface charge distributions, indicated that these industrially pure strains had undergone substantial variation. Treatment of the flocculent cells with pronase tended to reduce cell-to-cell contacts.
In the summary chapter the cell floe model was employed to describe the rheological behaviour of the yeast suspensions. Estimation of the force needed to separate doublet yeast cells were made using critical shear rate data (i.e., the point at which Bingham flow begins). This estimate was similar to that reported for single antibody bonds and may be due to the presence of lectin-like structures on the yeast cell wall.<br>Land and Food Systems, Faculty of<br>Graduate
14
Yip, Hopi. "Genetic manipulation of baker's yeast for improved maltose utilisation." Thesis, [Richmond, N.S.W.] : Centre for Biostructural and Biomolecular Resarch, Faculty of Science and Technolocy, University of Western Sydney, Hawkesbury, 1999. http://handle.uws.edu.au:8081/1959.7/223.
Testo completoAbstract (sommario):
Two yeast/E.coli shuttle vector plasmids were studied in 1994, termed pIBIDB and pBP33. According to this study, each plasmid should contain at least one ADH2UAS (upstream activation sequence in the alcohol dehydrogenase 2 gene) insert. In the present study, the constructed plasmids were analysed and transformed into laboratory strain yeast. The aim of this project was to identify the orientation, quantity and quality of the insert in the selected plasmids. Methods such as restriction analysis, polymerase chained reaction (PCR), sequencing, plate assays and enzyme assays were used to identify and evaluate the novel inserts. The data presented in this thesis suggest the inserted ADH2UAS fragment did enhance the production of maltose permease and maltase when the transformants were cultivated in maltose and ethanol-glycerol medium. The results suggested that transformants containing two inserts of ADH2UAS had a greater influence on the transformants than a single insert. But the inserts within the vectors and in transformed laboratory stain yeast appeared unstable. This could be due to the method used for plasmid construction and the storage condition of the transformants
15
Govender, Patrick. "Industrial yeast strains engineered for controlled flocculation." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/1450.
Testo completoAbstract (sommario):
Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009.<br>In many industrial fermentation processes, Saccharomyces cerevisiae yeast should ideally meet two partially conflicting demands. During fermentation a high suspended yeast count is of paramount importance to maintain a rapid fermentation rate, whilst efficient flocculation should ideally be initiated only on completion of the primary alcoholic fermentation, so as to enhance product clarification and recovery. Most commercial wine yeast strains are non-flocculent, probably because this trait was counter-selected to avoid fermentation problems. In this study, we assessed molecular strategies to optimise the flocculation behaviour of non-flocculent laboratory and wine yeast strains. For this purpose, the chromosomal copies of three dominant flocculation genes, FLO1, FLO5 and FLO11, of a non-flocculent S. cerevisiae laboratory strain (FY23) and two commercial wine yeast strains (BM45 and VIN13) were placed under the transcriptional control of the stationary phase-inducible promoters of the S. cerevisiae ADH2 or HSP30 genes.
Under standard laboratory media and culture conditions, all six promoter-gene combinations resulted in specific flocculation behaviours in terms of timing and intensity. The data show that the strategy resulted in the expected and stable expression patterns of these genes in both laboratory and industrial wine yeast strains. Most importantly, the data confirm that inducible expression of the native FLO1 and FLO5 open reading frames, albeit to varying degrees, are responsible for a quantifiable cell-cell adhesion phenotype that can be characterized as a Flo1 flocculation phenotype. On the other hand, we found that inducible expression of the native FLO11 ORF under these conditions resulted in flor/biofilm formation and invasive growth phenotypes. However, the specific impact of the expression of individual dominant FLO genes with regard to characteristics such as flocculation efficiency, cell wall hydrophobicity, biofilm formation and substrate adhesion properties showed significant differences between the commercial strains as well as between commercial and laboratory strains. These adhesion phenotype differences may at least in part be attributed to wine yeast FLO gene open reading frames containing significantly smaller intragenic repeat regions than laboratory strains.
The data show that the ADH2 regulatory sequences employed in this study were unsuitable for the purpose of driving FLO gene expression under wine-making conditions. However, HSP30p-based FLO1 and FLO5 wine yeast transformants displayed similar flocculent phenotypes under both synthetic and authentic red wine-making conditions, and the intensities of these phenotypes were closely aligned to those observed under nutrient-rich YEPD conditions. The fermentation activities of
HSP30p-based transgenic yeast strains were indistinguishable from that of their parental host wine yeast strains. The chemical composition of wines obtained using transgenic yeast strains were similar to those produced by parental strains. The BM45-derived HSP30p-FLO5 transformant in particular was capable of generating compacted or ‘caked’ lees fractions, thereby providing a distinct separation of the fermented wine product and lees fractions. Furthermore, in this study we report a novel FLO11 induced flocculation phenotype that seems to exclusively develop under authentic red wine-making conditions. This strong FLO11 flocculation phenotype was not wine yeast strain dependant, possessed both Ca2+-dependant and Ca2+-independent flocculation characteristics and was insensitive to inhibition by both glucose and mannose. A distinct advantage of this unique FLO11 phenotype was highlighted in its ability to dramatically promote faster lees settling rates. Moreover, wines produced by HSP30p-FLO11 wine yeast transformants were significantly less turbid than those produced by their wild type parental strains. The benefit of this attractive property is it facilitates simpler and faster recovery of wines and also promotes greater volume recovery of the wine product.
16
Niebauer, Ronald Thomas. "Engineering yeast cells for optimal expression of the human adenosine (A2a) receptor." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 4.52 Mb., 175 p, 2005. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3181857.
Testo completo
17
Crimmins, Kay. "The significance of genetic regulation in the control of glycolysis in Saccharomyces cerevisiae." Thesis, University of Aberdeen, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320258.
Testo completoAbstract (sommario):
The aim of this work was to establish the relative contribution of genetic regulation of the <I>PYK1</I>, <I>PFK1</I> and <I>PFK2</I> genes to the control of glycolysis. A series of isogenic mutant strains were constructed where the promoters and 5' untranslated sequences of the <I>PYK1, PFK1</I> and <I>PFK2</I> genes were replaced with those from <I>PGK1</I>. In addition , a second series of mutant strains were constructed where synthesis of Pyk1p and Pflkp was driven by the <I>PGK1<sub>Δuas</sub></I> promoter. These latter series of mutants were designed to contain weak expression of Pyklp and Pflkp. Analysis of UKC1 (<I>PGK::PYK1)</I> in shake flask cultures revealed similar growth rates on glucose and on lactate and similar rates of ethanol production and glucose consumption to those of the wild-type strain. This suggested that the native genetic regulation did not appear to play a significant role in the control of glycolysis. Nonetheless, analysis of this strain in the fermentor revealed that genetic regulation of <I>PYK1</I> may be important in co-ordinating Pyk1p synthesis, under the conditions studied. Analysis of YKC11 (<I>PGK<sub>Δuas</sub>::PYK1)</I> in both shake flask and fermentor experiments showed that genetic control was important in maintaining Pyk1p levels in order to sustain glycolytic flux. Shake flask analysis of the single and double <I>PFK</I> mutants under the control of the <I>PGK1</I> promoter revealed that the genetic regulation of the <I>PFK1</I> and <I>PFK2</I> genes did not appear to be important in the control of glycolysis. Weak expression of the <I>PFK1</I> and <I>PFK2</I> genes, under the control of the <I>PGK<sub>Δuas</sub></I> promoter showed the importance of genetic regulation in maintaining Pflkp levels to support glycolytic flux, under the conditions studied.
18
Bowen, Suzanne. "Stress and stationary phase characteristics in cell wall defective strains of Saccharomyces cerevisiae." Thesis, University of Bath, 2000. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341199.
Testo completo
19
Campos, Katherine Helen de Sa. "Identification and characterization of components that overcome secretion limitations of the yeast Pichia pastoris." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/844.
Testo completoAbstract (sommario):
The methylotrophic yeast. Pichia pastoris, is a powerful, adaptable, and inexpensive recombinant expression system commonly used to secrete heterologous protein. Although P. pastoris is a popular host organism, secretion inefficiency continues to be a major hurdle in its ability to produce high levels of foreign protein. Optimization of cis- and trans-acting factors has greatly enhanced the secretory capabilities of P. pastoris, however protein-specific engineering of a host organism is costly and not always effective. P. pastoris' secretion inefficiency is commonly due to trans-acting factors. Strains of S. cerevisiae have been engineered, through random genomic mutation, that are capable of overcoming these /ram-acting factors to secrete high levels of foreign protein. The Lin-Cereghino laboratory at University of the Pacific has developed a screen to identify mutations in P. pastoris capable of circumventing secretion obstacles. The P. pastoris genome was randomly disrupted through restriction enzyme-mediated integration of an antibiotic resistance marker. Supersecretion mutants were identified by their ability to secrete β-galactosidase, a reporter enzyme not natively secreted by P. pastoris. Sixteen β-galactosidase secretion (bgs) mutants were initially isolated by the Lin-Cereghino lab. This research focused on characterizing one of the resultant bgs mutants, ///. Initial sequencing and alignment studies identified the predicted LI1p sequence to be homologous to S. cerevisiae protein kinase C (PKC). Considering the role of PKC in the Cell Wall Integrity pathway of S. cerevisiae. the cell wall and secretory organelles of III were closely examined using transmission electron microscopy. Additionally, a qualitative alkaline phosphatase assay was used to evaluate the cell wall integrity of ///. Finally, the secretory phenotype of 111 was examined using a group of structurally and functionally diverse reporter proteins. In characterizing the bgs mutant, III, this research contributes to an understanding of cellular components that limit protein secretion in the yeast, P. pastoris.
20
Boeira, Lucia Schuch. "Effects of fusariotoxins on the performance of brewing yeast strains." Thesis, Heriot-Watt University, 2000. http://hdl.handle.net/10399/560.
Testo completo
21
La, Grange-Nel Karin. "Characterisation and improvement of whiskey yeast." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53327.
Testo completoAbstract (sommario):
Thesis (MSc)--Stellenbosch University, 2003.<br>ENGLISH ABSTRACT: Scotch whiskey is of two main types, namely Scotch malt whiskey, made from malted
barley alone, or Scotch grain whiskey, made from cereals, such as wheat or maize,
together with malted barley. In both processes, the enzymes from the barley are
responsible for starch conversion and should always be derived entirely from the
malted barley. No exogenous enzymes are allowed to be added to any mashing.
The enzymes involved in the conversion process to fermentable sugars, are the aand
p-amylases, limit dextrinase and p-glucosidase.
Maize, on the other hand, contains no enzyme activity, therefore enzymes need
to be added when producing whiskey from maize alone. In other whiskey-producing
countries where maize is freely available and cheaper than barley, the use of
exogenous enzymes are allowed in the mashing process and is crucial for the
formation of fermentable sugars from complex carbohydrates. The cost of the
enzymes, however, can push the production cost of whiskey to higher levels.
Saccharomyces cerevisiae does not have any amylolytic activity, but is an
excellent fermenter and produces favourable organoleptic notes, which makes it very
suitable for producing potable spirit. Efforts have been made to genetically improve
industrial strains, relying on classical genetic techniques followed by the selection of
broad traits, such as ethanol tolerance, absence of off-flavours and
carbohydrate/starch utilisation. No strain has thus far been selected for total starch
degradation during the fermentation of whiskey mash.
Over the last decade, considerable progress has been made in the development
of genetically improved strains for the distilling, wine, brewing and baking industries.
The expression of heterologous genes introduced a new dimension in approaches to
the genetic improvement of industrial strains. It would therefore be cost-effective to
use a yeast strain that can produce active and sufficient enzymes to ferment raw
starch efficiently to alcohol without lowering the quality of the end product. No such
strain has been developed to date, but the continuous improvement of starch-utilising
strains has made this goal more achievable.
Two a-amylase genes, namely LKA 1 and LKA2, were previously isolated from
Lipomyces kanonenkoae. In this study, we selected 4 strains on the basis of criteria
that are important for whiskey-specific strains. The selected strains were
transformed with LKA 1, as well as with a combination of LKA 1 and LKA2 genes. The
wine yeast VIN13 was included in the transformation of LKA1 and LKA2 because of
its rapid fermentation rate. The genes were integrated into the genomes of the yeast
strains and were stable after many generations. Assays showed that a significant
increase in enzyme activity was induced in the whiskey strains, compared to the
untransformed strains. The strains also showed good fermentation ability in whiskey
fermentations, although optimum alcohol production was still not achieved.<br>AFRIKAANSE OPSOMMING: Skotse whiskey bestaan uit 2 tipes, nl. mout whiskey, gemaak slegs van mout d.w.s.
gars wat die mout proses ondergaan het, en graan whiskey wat gemaak word van
gewasse soos mielies of koring, waarby mout gevoeg word. Die ensieme afkomstig
van die mout is verantwoordelik vir die omsetting van stysel na fermenteerbare
suikers en geen eksogene ensieme mag by die gars- of graanmengsel gevoeg word
nie. Die ensieme wat betrokke is by die omsetting van stysel, is die a- en ~-
arnitases, limiet dekstrinase en ~-glukosidase.
Mielies bevat geen ensiemaktiwiteit nie, dus moet ensieme by die proses gevoeg
word indien slegs mielies vir die vervaardiging van whiskey gebruik word. In whiskey
produserende lande waar mielies vryelik beskikbaar is en goedkoper is as gars, word
eksogene ensieme by die graanmengsel gevoeg vir die vrystelling van
fermenteerbare suikers vanaf komplekse koolhidrate. Die hoë koste van die ensieme
kan egter die produksiekoste van whiskey verhoog.
Saccharomyces cerevisiae besit geen amilolitiese aktiwiteit nie, maar is 'n
uitstekende fermenteerder en produseer gewensde organoleptiese geure. Om
hierdie redes is S. cerevisiae baie geskik vir die produksie van drinkbare etanol.
Navorsingspogings om industriële rasse geneties m.b.v. klassieke genetiese
metodes te verbeter, kom wydverspreid in die literatuur voor. Dit sluit in die seleksie
van rasse met 'n verskeidenheid van eienskappe soos etanol toleransie, die
afwesigheid van afgeur produksie en koolhidraat/stysel benutting. Geen ras is egter
tot op hede geselekteer vir totale stysel afbraak gedurende fermentasie nie.
Groot vordering is gedurende die laaste dekade gemaak in die ontwikkeling van
genetiese verbeterde rasse vir die wyn- stokery- en brouers industrieë. Die uitdruk
van heterogene gene in gisrasse gee 'n nuwe dimensie aan die genetiese
verbetering van industriële rasse. Die gebruik van 'n gisras wat aktiewe en
genoegsame ensieme produseer om rou stysel te fermenteer, sonder om die kwalitiet
van die eindproduk nadelig te beïnvloed, kan die produksiekoste van whiskey
aansienlik verminder. Geen gisras met hierdie eienskap is tot op hede ontwikkel nie,
maar die voortdurende verbetering van rasse om stysel af te breek maak hierdie doel
meer bereikbaar.
Twee a-amilase gene, nl. LKA 1 en LKA2 is voorheen uit Lipomyces
kononenkoae geïsoleer. In hierdie studie is 4 gisrasse geselekteer op grond van die
kriteria wat nodig is vir whiskey giste. Die geselekteerde rasse is getransformeer met
LKA 1 sowel as 'n kombinasie van LKA 1 en LKA2 gene. Die wyngis VIN13 is
ingesluit by die transformasie met die LKA1 en LKA2 gene, omrede VIN13 bekend is
as 'n vinnige fermenteerder. Die gene is geïntegreer in die genoom van die
verskillende gisrasse en is stabiel na vele generasies. Die getransformeerde rasse
het 'n betekenisvolle verhoging in ensiemaktiwiteit teenoor die nie-getransformeerde rasse getoon. AI die transformante het ook goeie fermentasie vermoë getoon in
whiskey fermentasie proewe. Optimum alkoholproduksie is egter nie verkry nie.
22
Yip, Hopi, of Western Sydney Hawkesbury University, and Faculty of Science and Technology. "Genetic manipulation of baker's yeast for improved maltose utilisation." THESIS_FSTA_SFS_Yip_H.xml, 1999. http://handle.uws.edu.au:8081/1959.7/223.
Testo completoAbstract (sommario):
Two yeast/E.coli shuttle vector plasmids were studied in 1994, termed pIBIDB and pBP33. According to this study, each plasmid should contain at least one ADH2UAS (upstream activation sequence in the alcohol dehydrogenase 2 gene) insert. In the present study, the constructed plasmids were analysed and transformed into laboratory strain yeast. The aim of this project was to identify the orientation, quantity and quality of the insert in the selected plasmids. Methods such as restriction analysis, polymerase chained reaction (PCR), sequencing, plate assays and enzyme assays were used to identify and evaluate the novel inserts. The data presented in this thesis suggest the inserted ADH2UAS fragment did enhance the production of maltose permease and maltase when the transformants were cultivated in maltose and ethanol-glycerol medium. The results suggested that transformants containing two inserts of ADH2UAS had a greater influence on the transformants than a single insert. But the inserts within the vectors and in transformed laboratory stain yeast appeared unstable. This could be due to the method used for plasmid construction and the storage condition of the transformants<br>Master of Science (Hons)
23
Boynton, Primrose. "Ecological Patterns and Processes in Sarracenia Carnivorous Pitcher Plant Fungi." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10636.
Testo completoAbstract (sommario):
The kingdom Fungi is taxonomically and ecologically diverse, containing an estimated 1.5 million species. Fungi include decomposers, pathogens, and plant and animal mutualists. Many fungi are microorganisms, and the processes shaping microbial diversity may be fundamentally different from those that shape plants and animals. However, ecologists do not yet fully understand how fungal species are distributed over space and time. Using fungi that inhabit the water of Sarracenia carnivorous pitchers, I describe inter and intraspecific fungal diversity and investigate the processes that shape fungal diversity. I introduce these concepts in Chapter 1. In Chapter 2, I describe changes in fungal species diversity over space and time. I enumerated fungal species in five Sarracenia populations across the United States and Canada, and show that thousands, but not hundreds of kilometers separate distinct fungal communities. I also sampled a single Sarracenia population over a Sarracenia growing season, and found that young fungal communities are significantly different from older fungal communities. Observed patterns correlate with environmental factors including temperature and pitcher pH, and with the presence or population structure of pitcher inhabiting arthropods. In Chapter 3, I describe dispersal of and competition among three common pitcher fungi. I tracked Candida glaebosa, Rhodotorula glutinis, and Pseudozyma aphidis appearances in pitchers in a single Sarracenia population, and show that different appearances reflect different dispersal times. I also describe interactions between dispersal and competition in microcosms: high numbers of propagules introduced into a microcosm give a competitive advantage to investigated fungi. In Chapter 4, I describe changes in genotype composition of a population of Candida glaebosa, which is widespread and abundant in pitchers, and disperses early in the season. I observed three C. glaebosa populations in five locations; C. glaebosa population structure does not reflect broader community structure as described in Chapter 2. Population structure instead correlates with host taxonomy, and I contrast inter and intraspecific diversity patterns and the processes that potentially cause such patterns.
24
Stanislaw, Justina Marie. "Identification and analyzation of a gene preferentially expressed in the yeast phase of thepathogenic fungus Talaromyces marneffei." Youngstown State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1596025620277104.
Testo completo
25
Rizzi, John. "Production of emulsifier by Torulopsis petrophilum." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=64014.
Testo completo
26
Ho, Hei Ngam Jennifer. "Functional characterization of yeast NMD3 in the biogenesis and transport of the large (60S) ribosomal subunit /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004287.
Testo completo
27
Zhao, Jianming 1972. "Classification and identification of yeasts by Fourier transform infrared spectroscopy." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=31564.
Testo completoAbstract (sommario):
Infrared spectra of microbial cells are highly specific, fingerprint-like signatures which can be used to differentiate microbial species and strains from each other. In this study, the potential applicability of Fourier transform infrared (FTIR) spectroscopy for the classification of yeast strains in terms of their biological taxonomy, their use in the production of wine, beer, and bread, and their sensitivity to killer yeast strains was investigated. Sample preparation, spectral data preprocessing methods and spectral classification techniques were also investigated. All yeast strains were grown on a single growth medium. The FTIR spectra were baseline corrected and the second derivative spectra were computed and employed in spectral analysis. The classification accuracy was improved when the principal component spectra (calculated from the second derivative spectra) were employed rather than the second derivative spectra or raw spectra alone. Artificial neural network (ANN) with 10 units in the input layer and 12 units in the hidden layer produced a robust prediction model for the identification of yeasts. Cluster analysis was employed for the classification of yeast strains in terms of their use in the production of wine, beer, and bread and in terms of their sensitivity to killer yeast strains. The optimum region for the classification in the former case was found to be between 1300 and 800 cm-1 in the infrared spectrum whereas the optimum region for the classification of yeast strains in terms of their sensitivity was between 900 and 800 cm-1 . The results of this work demonstrated that FTIR spectroscopy could be successfully employed for the classification and identification of yeast strains with minimal sample preparation.
28
Smit, Annel. "Maltotriose transport in yeast." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/21760.
Testo completoAbstract (sommario):
Dissertation (PhD)--University of Stellenbosch, 2007.<br>ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been
intertwined with human culture and long as civilized man has existed. This fermentation
process has been dominated by the micro-organism Saccharomyces cerevisiae and from
providing ancient seafaring explorers of a non perishable beverage to equipping bakers
with a raising agent to turn flour into bread; this organism with its fermentative potential,
has formed an essential part of most societies.
In more recent times, many industries still rely on this basic principle. The
complexities and efficiencies of the conversion of sugar into its various fermentative byproducts
have been studied and optimised extensively to meet the specific demands of
industries. Depending on the raw material used as starting point, the major beneficiaries of
the useful characteristics have been alcoholic beverage producers (wine, beer, and
whiskey amongst others), bakers (bread leavening) and biofuel producers.
One of the obstacles in fermentation optimisation is the sugar consumption
preferences displayed by the organism used. S. cerevisiae can consume a wide variety of
sugars. Depending on the complexities of its structures, it shows a preference for the
simpler saccharides. The fermentation of certain more complex sugars is delayed and runs
the risk of being left residually after fermentation. Many of the crops utilised in
fermentation-based products contain large amounts of starch. During the starch
degradation process many different forms of sugars are made available for fermentation.
Improved fermentation of starch and its dextrin products would benefit the brewing,
whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and
maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin
products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to
transport the aforementioned mono-, di- and trisaccharides into the cytosol. The
inefficiency of maltotriose transport has been identified as the main cause for residual
maltotriose. The maltotriose transporting efficiency also varies between different
Saccharomyces strains.
By advancing the understanding of maltotriose transport in yeast, efforts can be
made to minimise incomplete fermentation. This aim can be reached by investigating the
existing transporters in the yeast cell membrane that show affinity for maltotriose. This
study focuses on optimising maltotriose transport through the comparison of the alpha
glucoside transporter obtained from different strains of Saccharomyces. Through specific
genetic manipulations the areas important for maltotriose transport could be identified and
characterised.
This study offers prospects for the development of yeast strains with improved maltose
and maltotriose uptake capabilities that, in turn, could increase the overall fermentation
efficiencies in the beer, whiskey, and biofuel industries.<br>AFRIKAANSE OPSOMMING: Die transformasie van suiker na etanol en koolstof dioksied is so oud soos die beskawing self, en dit is van die vroegste tye af onlosmaaklik met die mens se kultuur verbind. Hierdie fermentasie-proses word gedomineer deur die Saccharomyces cerevisiae mikroorganisme. Hierdie organisme het antieke seevaarders voorsien van ‘n nie-bederfbare drankie en van ouds af aan bakkers ‘n rysmiddel verskaf waarmee meel in brood verander kon word. As gevolg van hierdie fermenteringspotensiaal het hierdie organisme ‘n onmisbare rol in meeste beskawings gespeel. Baie industrieë is steeds op hierdie basiese beginsel gebou. Die kompleksiteite en effektiwiteit van die transformasie van suiker na sy verskeie gefermeenteerde neweprodukte is breedvoerig bestudeer en geoptimiseer om aan die spesifieke behoeftes van verskeie industrieë te voeldoen. Afhangend van die grondstowwe wat as beginpunt gebruik is, is die primêre begunstigdes van die fermentasie proses die alkoholiese drankprodusente (onder andere die wyn-, bier- en whiskey produsente), bakkers en biobrandstofprodusente. Die suikerverbruik-voorkeur van die organisme wat die fermentering fasiliteer is een van die struikelblokke in die optimisering van die proses. S. cerevisiae kan ‘n wye spektrum van suikers verbruik maar dit toon ‘n voorkeur vir die eenvoudiger suikers. Die fermentasie van sekere van die meer komplekse suikers is vertraag en loop die risiko om agtergelaat te word na fermentasie. Vele van die gewasse wat in die gefermenteerde produkte gebruik word bevat groot hoeveelhede stysel. Vele soorte suikers word gedurende die afbreek van die stysel beskikbaar gestel vir fermentasie. Die brouers-, whiskey- en biobrandstof industrieë sal almal voordeel trek uit die verbeterde fermentasie van stysel en sy gepaardgaande dekstrin produkte. Meeste Saccharomyces gisrasse fermenteer glucose en maltose; maltotriose word gedeeltelik gefermenteer, maar die meer komplekse dekstrien produkte gevind in stysel word nie gefermenteer nie. Hierdie verbruikerspatroon kan gedeeltelik toegeskryf word aan die vermoë van gisselle om die bogenoemde mono-, di- and trisaccharides in die sitosol op te neem. Die oneffektiwiteit van maltotriose transport is identifiseer as die hoofoorsaak van post-fermentatiewe, oortollige maltotriose. Die effektiwiteit van maltotriose transport verskil ook tussen verskillende Saccharomyces rasse. Pogings om onvolledige fermentasie te veminder kan bevorder word deur die kennis rondom maltotriose transport in gis uit te bou. Hierdie oogmerk kan bereik word deur die bestaande transporters in die gissel se membraan wat ‘n affiniteit vir maltotriose toon te ondersoek. Hierdie studie fokus op die optimisering van maltotriose transport deur die vergelyking van die alpha glucoside transporter (AGT1) wat van verskillende Saccharomyces rasse afkomstig is. Die areas wat relevant is tot maltotriose transport kon deur spesifieke genetiese manipulasies identifiseer en gekarakteriseer word. Hierdie studie bevorder die vooruitsig op die ontwikkeling van gisrasse met verbeterde
maltose en maltotriose transport vermoëns wat op sy beurt weer kan aanleiding gee tot die
verbeterde fermentasie effektiwiteit in die bier, whiskey en biobrandstof industrieë.
29
You, Tao. "Modelling and simulation of amino acid starvation responses in yeast Saccharomyces cerevisiae." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources. Restricted: no access until June 2, 2014, 2009. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=25979.
Testo completo
30
Al-Talhi, Abdullah Dakheel D. "Environmental and medical studies on microbial growth under low nutrient (oligotrophic) conditions." Thesis, University of Sheffield, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340132.
Testo completo
31
Davey, Claire Louise. "Reductions of aromatic carboxylic acids and nitroarenes using whole cell biotransformations." Thesis, University of Exeter, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361337.
Testo completo
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Schoeman, Heidi. "The fate of genetically modified yeast in the environment." Thesis, Stellenbosch : Stellenbosch University, 2005. http://hdl.handle.net/10019.1/50489.
Testo completoAbstract (sommario):
Dissertation (PhD(Agric))--University of Stellenbosch, 2005.<br>ENGLISH ABSTRACT: Considerable efforts have been made to improve strains of the wine yeast
Saccharomyces cerevisiae through the use of genetic engineering. Although the
process is well defined, globally there is much resistance towards the use of
genetically modified organisms (GMOs), primarily because little is known about their
environmental fate and their potential effect on naturally occurring organisms. The
public concern is mainly focused on the uncertainty associated with the impact of the
deliberate or accidental release of a GMO into the environment. As a consequence,
thére is an urgent need to assess the potential risks involved with the use of this new
technology. For the eventual global acceptance of any GMO, it is imperative that the
consumer must be convinced that it is ultimately safe for human consumption and the
environment. In order to achieve this, certain risk assessment procedures must be
performed on each and every GMO that is planned to be released into the
environment. Although some of the genetically modified (GM) yeasts that have been
developed comply with the strict legislation of most countries and have been cleared
by regulatory authorities for commercial use, GM yeasts have not, as yet, been used
for the commercial production of GM bread, beer or wine. Nevertheless, the use of
GM yeasts in the market appears imminent and there is an urgent need to assess
and address the perceived health and environmental risks associated with GM foods.
The overall objective of this research was to evaluate key environmental issues
concerning the use of GM yeasts. The focus was on comparing the behaviour of
specific parental and GM yeast strains in model systems in order to determine
whether the GM strains may have any selective advantage, which could lead to their
spreading. Specifically, it involved monitoring of the growth behaviour of selected
GM yeasts within a vineyard microbial community and in fermentations, as well as
the interaction of these yeasts with sand and glass surfaces in an aqueous
environment. The GM yeasts under investigation were recombinant strains of a
well-known, industrial strain of S. cerevisiae VIN13 expressing an a-amylase
(designated GMY1); an endo-p-1,4-glucanase and endo-p-xylanase (designated
GMY2); and a pectate lyase and polygalacturonase (designated GMY3).
The GM yeasts were mist-inoculated onto individually-contained blocks
consisting of one-year old grapevines in a secluded glasshouse environment.
Specifically, the numbers and dynamics of GM yeast survival, as well as the effect of
an introduced GM yeast on the yeast community dynamics and numbers, were
investigated. Overall, it was found that the most prevalent wild yeasts isolated from
the grapevines were Rhodo torula, Yarrowia lipolytica, Pichia and Candida spp.
VIN13 and the GM yeasts did not affect the overall ecological balance of the
microflora on the grapevines. Wild strains of S. cerevisiae were seldom isolated from
the grapevines. With a few exceptions, the overall detection of GM yeasts was
numerically limited. Co-inoculation of (VIN13+GMY1) and (GMY1+GMY2) revealed detection approximately in the same ratio at which they were inoculated, with small
differences in the order of GMY2>GMY1 >GMY3. GM yeasts were rarely isolated
from bark and soil samples. Spontaneous fermentation of the grapes harvested from
the different treated blocks indicated that the GM yeasts survived on the berries, that
the natural fermenting ability of VIN13 was conserved in the recombinant strains, and
that the GM yeasts did not have any competitive advantage.
The soil environment forms an important part of the biosphere and the transport
and attenuation of a GM yeast in this matrix will to a large extent affect their ultimate
fate in the environment. In soil, microorganisms either occur as suspended cells in
pore water or as biofilms on soil surfaces. Although less extensive than a typical soil
yeast, Cryptococcus, epifluorescent staining of biofilms confirmed that VIN13 and
GMY1 were capable of existing in a biofilm mode on sand granules and glass. Data
on effluent numbers detected in flow cells indicated that GMY1 had no advantage
due to the genetic modification and had the same reproductive success as VIN13.
These strains either had no difference in biofilm density or GMY1 was less dense
than VIN13. When co-inoculated, GMY1 had no negative influence on the mobility of
Cryptococcus through a sand column, as well as the ability of Cryptococcus to form
biofilms. Furthermore, it was found that GMY1 did not incorporate well into a stable
biofilm community on glass, but did not disrupt the biofilm community either.
This is the first report of the assessment of the fate of GM strains of VIN13 that
are suitable for the wine and baking industry. The investigation of the GM yeasts in
this study under different scenarios is a good start to an extensive and necessary risk
assessment procedure for the possible use of these GM yeasts in the industry. This
study could lead to the provision of much-needed scientific and technical information
to both industry and regulating bodies. The outcome of this research is also intended
to serve as a basis for information sharing with public interest groups.<br>AFRIKAANSE OPSOMMING: Aansienlike pogings is reeds aangewend om rasse van die wyngis, Saccharomyces
cerevisiae, deur middel van genetiese manipulering te verbeter. Alhoewel hierdie
proses goed gedefinieerd is, is daar wêreldwyd heelwat teenkanting teen die gebruik
van geneties gemanipuleerde organismes (GMO's). Dit is hoofsaaklik te wyte
daaraan dat so min bekend is oor hul lot in die omgewing en hul potensiële effek op
die organismes wat natuurlik voorkom. Die publiek is veral besorg oor die
onsekerheid verbonde aan die bestemde of toevallige vrylating van 'n GMO in die
omgewing. Gevolglik is daar 'n dringende behoefte om die potensiële risiko's in die
gebruik van hierdie nuwe tegnologie te bepaal. Dit is van uiterste belang dat die
verbruiker oortuig sal word van die veiligheid vir menslike gebruik en die omgewing
voordat enige GMO uiteindelik wêreldwyd aanvaarbaar sal word. Om dit te kan
bereik sal sekere risiko-bepalende prosedures toegepas moet word op ieder en elke
GMO wat beplan word om vry gelaat te word in die omgewing. Alhoewel sommige
van die geneties gemanipuleerde (GM) giste aan die streng wetgewing van die
meeste lande voldoen en deur die owerhede vir kommersiële gebruik goedgekeur is,
word GM-giste nog steeds nie vir die produksie van GM brood, bier of wyn gebruik
nie. Ten spyte hiervan, blyk die gebruik van GM-giste onafwendbaar te wees en is
daar dus 'n dringende behoefte om die voorspelde gesondheids- en
omgewingsrisiko's wat met GM voedsel geassosieer word, aan te spreek.
Die oorhoofse doel van hierdie navorsing was om belangrike
omgewingskwessies aangaande die gebruik van GM-giste te evalueer. Die fokus
was op die vergelyking van die gedrag van spesifieke oorspronklike gisrasse en
GM-gisrasse in modelsisteme sodat daar bepaal kon word of die GM-gisrasse enige
selektiewe voordele het wat moontlik tot hulonbeheerde verspreiding in die natuur
sou kon lei. Dit het spesifiek die monitering van die groei van geselekteerde GMgiste
binne 'n mikrobiese gemeenskap op wingerd en in fermentasies behels, asook
die interaksie van hierdie giste met grond en glas oppervlaktes in 'n wateromgewing.
Die GM-giste wat in hierdie studie gebruik is, was rekombinante rasse van 'n
bekende industriële ras van S. cerevisiae, VIN13, wat geneties gemodifiseerd was
om 'n a-amylase (aangedui as GMG1); 'n endo-p-1,4-glukanase en 'n
endo-B-xilanase (aangedui as GMG2); en 'n pektaatliase en 'n poligalaktorinase
(aangedui as GMG3) uit te druk. Die GM-giste is op afsonderlike blokke van
eenjaaroue wingerdplante binne-in 'n beskutte kweekhuis gesproei-inokuleer. Daar
was spesifiek na die selgetalle en dinamika van die oorlewende GM-giste gelet,
asook wat die invloed was van die inokulasie van 'n GM gis op die selgetalle van die
natuurlike gisgemeenskap. Daar is bevind dat die wildegiste Rhodotorula, Yarrowia
Iipolytica, Pichia en Candida spp die gereeldste van die wingerd geïsoleer is. VIN13
en die GM-giste het nie die ekologiese balans van die natuurlike mikrobiese
populasie op die wingerd versteur nie. Wilde rasse van S. cerevisiae is selde
geïsoleer vanaf die wingerd. In die meeste gevalle is daar bevind dat wanneer GM-giste opgespoor is, hulle in lae selgetalle voorgekom het. Waar giste saam
geïnokuleer was, was die opsporing van (VIN 13+GMY1) en (GMY1 +GMY2)
ongeveer in dieselfde verhouding as waarin hul geïnokuleer was, terwyl klein
verskille in die orde van GMY2>GMY1 >GMY3 opgemerk is. GM-giste is selde vanaf
bas- en grond-monsters geïsoleer. Spontane fermentasies van druiwe wat geoes
vanaf die verskillende behandelde blokke is, het daarop gedui dat die GM-giste wel
op die druiwe oorleef, dat die natuurlike vermoë van VIN13 om te kan fermenteer in
die gemodifiseerde gisrasse behoue gebly het en dat die GM-giste geensins deur die
genetiese modifikasies bevoordeel was nie.
Grond is 'n belangrike deel van die biosfeer en die verspreiding en aanhegting
van 'n GM-gis in hierdie matriks sal sy algehele lot in die omgewing tot 'n groot mate
beïnvloed. In die grond kom mikroorganismes as gesuspendeerde selle in
poriewater of as biofilms op die oppervlaktes van grond voor. Alhoewel
biofilmvorming van VIN13 en GMG1 swakker was as in die geval van 'n tipiese
grondgis, Cryptococcus, het epifluoresserende kleuring van hierdie S. cerevisiaegiste
bevestig dat VIN13 en GMG1 in staat was om as biofilms op sandkorrels en
glas te oorleef. Gebaseer op seltellings in vloeiseluitlaat, kon daar afgelei word dat
GMG1 geen selektiewe voordeel geniet het as gevolg van die genetiese modifikasie
nie en dat die gis net so reproduktief was as VIN13. Hierdie gisrasse het geen
verskil in biofilmdigtheid getoon nie of die biofilmvorming van GMG1 was minder dig
as die van VIN13. Wanneer GMG1 saam met Cryptococcus geïnokuleer was, het
GMG1 geen negatiewe invloed op die beweeglikheid van Cryptococcus deur 'n
sandkolom gehad nie en die vermoë van Cryptococcus om biofilms te vorm is ook nie
beïnvloed nie. Daar is verder ook bevind dat GMG1 nie goed binne-in 'n
gestabiliseerde biofilmgemeenskap op glas geïnkorporeer het nie, maar dat die gis
ook nie die biofilmgemeenskap versteur het nie.
Hierdie studie verteenwoordig die eerste ondersoek ooit oor die lot, oorlewing en
groeigedrag van GM-wyngiste in biologies-afgesonderde wingerd-, fermentasie-,
modelgrond- en modelwater-ekosisteme. Die bestudering van hierdie GM-giste
onder verskillende omgewingstoestande in afgeslote ekosisteme lê 'n stewige basis
vir verdere ondersoeke en die ontwikkeling van omvattende en noodsaaklike
risikobepalingsprosedures betreffende die moontlike toekomstige gebruik van
GM-giste in die industrie. Hierdie studie baan die weg tot die verkryging van
noodsaaklike wetenskaplike en tegniese inligting oor die veiligheidsaspekte rakende
GM-wyngiste en dit kan van groot waarde vir die industrie, owerhede en
verbruikerspubliek wees.
33
Muller, Christo A. "Monitoring the spreading of commercial wine yeasts in the vineyard." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53505.
Testo completoAbstract (sommario):
Thesis (MSc)--Stellenbosch University, 2003.<br>Full text to be digitised and attached to bibliographic record.<br>ENGLISH ABSTRACT: Traditionally, wine has been produced by the spontaneous fermentation of grape
juice by yeast that originate from the grapes and winery equipment. Research has
shown that the population composition and dynamics of these yeasts and other
microorganisms are very complex. Kloeckera and its anamorph, Hanseniaspora,
dominate the yeast population found on the surfaces of grapes, although prevailing
Saccharomyces cerevisiae strains complete the fermentation process.
The yeast S. cerevisiae is an important factor contributing to the quality of wines
and, therefore, the improvement of wine yeasts receives considerable attention
worldwide. Apart from classical yeast breeding studies, genetic engineering and
recombinant DNA techniques are increasingly being used in strain development
research programmes. These techniques might enable the wine yeasts to produce
heterologous enzymes that degrade polysaccharides, convert malic acid to lactic
acid, increase glycerol production, release roam and flavour compounds, secrete
antimicrobial peptides, etc. The release of recombinant yeast strains (genetically
modified organisms, GMOs) is subject to statutory approval. Therefore, it is important
to answer several questions prior to the use of such genetically improved yeast in the
commercial production of wine. For example, will recombinant yeast strains be able
to multiply and spread in nature, and will this GMO be able to out-compete the
natural microflora because of its newly acquired genetic traits. Since existing
commercial wine yeasts are used in the abovementioned strain development
research, it is essential to determine already at this early stage to what extent these
wine yeast strains survive and spread in nature and to what extent they influence the
fermentations of the following vintages.
This study is divided into two sections. The aim of the first section is to sample a
representative number of yeast strains from various vineyards in different
climatological areas, mainly in the Western Cape, South Africa. These yeast strains
were identified mainly by electrophoretic karyotyping (contour-clamped homogenous
electric field electrophoresis; CHEF).
The second part of the study summarises the results obtained when Fourier
transform infrared (FT-NIR) spectroscopy was used to differentiate commercial wine
yeast strains. Sets of data, containing the spectra of the mostly used commercial
wine yeast strains, were constructed and used as a reference library. The spectra of
the isolated yeast strains were then compared to the reference dataset with specific
FT-NIR computer software using mathematical calculations.
In conclusion, the two methods used in conjunction with one another proved that
the commercial wine yeast strains do not easily disperse from the cellar into the
vineyard. The commercial wine yeast strains are also more likely to be found near
the cellar and the places where the grape skins are dumped. Therefore, should a
recombinant yeast strain be used in winemaking, it would not be dispersed into the
vineyard. It therefore appears that the commercial use of genetically improved yeast does not pose a high risk in terms of dominance of the indigenous microbial
population in the environment<br>AFRIKAANSE OPSOMMING: Wyn is tradisioneel gemaak deur die natuurlike gisting van druiwesap deur giste wat
op die druiwe en keldertoerusting voorkom. Navorsing het getoon dat die
samestelling en dinamika van die gispopulasie en ander mikro-organismes baie
kompleks is. Kloeckera en sy anamorf, Hanseniaspora, domineer die inheemse
gispopulasie op druiwedoppe, terwyl Saccharomyces cerevisiae in baie klein getalle
op die druiwedoppe voorkom, maar later die fermentasie oorheers en uiteindelik
voltooi.
Die gis S. cerevisiae speel 'n baie belangrike rol in die kwaliteit van wyn en
daarom geniet die verbetering van wyngiste wêreldwyd besondere aandag.
Benewens die klassieke gistelingstudies, word genetiese manipuleringstegnieke
toenemnd in navorsingsprojekte gebruik wat daarop gefokus is om wyngisrasse te
verbeter. Hierdie tegnieke mag die giste in staat stelom heteroloë ensieme te
produseer wat polisakkariedes afbreek, appelmelksuur afbreek, gliserolproduksie
verhoog, smaak- en geurkomponente vrystel, antimikrobiese peptiede afskei, ens.
Voordat sulke geneties gemanipuleerde giste het egter in kommersiële wynproduksie
gebruik sal kan word, is daar heelwat wetlike vereistes waaraan voldoen sal moet
word en vrae wat vooraf beantwoord sal moet word. Byvoorbeeld, sal die
rekombinante giste in staat wees om vinniger te vermeerder as gevolg van die nuwe
genetiese eienskappe en sodoende die natuurlike populasies onderdruk? Omdat
kommersiële wyngiste in bogenoemde gisverbeteringprogramme gebruik word, is dit
noodsaaklik om nou reeds die verspreiding van die kommersiële giste te monitor en
te bepaal hoe geredelik hulle in die natuur kan versprei en oorleef, en hoe hulle
wynfermentasies van die daaropvolgende jare beïnvloed.
Die studie is in twee gedeeltes verdeel. Die doel van die eerste gedeelte was om
'n verteenwoordigende aantal gisrasse uit die wingerde van 'n aantal wynplase in
verskillende klimaatstreke te isoleer, spesifiek in die Wes-Kaap, Suid-Afrika. Die
gisrasse was grotendeels deur elektroforetiese kariotipering (kontoer-geklampte
homogene elektriese veld; CHEF) geïdentifiseer.
Die tweede deel van die navorsing was gefokus op die onderskeiding tussen die
mees gebruikte kommersiële wyngiste met 'Fourier-Transform Near Infrared' (FTNIR)
spektroskopie. Eerstens is 'n stel data, bestaande uit die spektrum data oor die
kommersiële wyngiste opgestel om as 'n verwysingsbiblioteek te dien. Tweedens is
die spektrum van data oor die geïsoleerde giste onder presies dieselfde toestande
met die verwysingsbiblioteek vergelyk. Dié tegniek maak dit moontlik om tussen die
kommersiële wyngiste te onderskei.
As die twee metodes saam gebruik word vir identifikasie, kan die afleiding
gemaak word dat kommersiële wyngiste nie maklik vanaf die kelder na die wingerd
versprei nie. Die kommersiële wyngiste is ook meestal naby die kelder en die
dopstortingsterreine gevind. Sou 'n rekombinante gisras dus gebruik word om wyn te maak, sal dit nie maklik versprei nie. Die kommersiële gebruik van geneties
gemanipuleerde wyngiste behoort dus nie In groot omgewingsrisiko in te hou nie.
34
Yang, Hui. "Chromosome dynamics and chromosomal proteins in relation to apoptotic cell death in yeast." Laramie, Wyo. : University of Wyoming, 2008. http://proquest.umi.com/pqdweb?did=1594496261&sid=1&Fmt=2&clientId=18949&RQT=309&VName=PQD.
Testo completo
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Ngsee, Johnny Kuan. "Cassette mutagenic analysis of the signal peptide of yeast invertase." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/27500.
Testo completoAbstract (sommario):
The SUC2 locus of Saccharomyces cerevisiae encodes two forms of invertase; a constitutively expressed cytoplasmic enzyme and a glucose-repressible secreted and glycosylated enzyme which is initially produced with an amino-terminal signal peptide. The coding sequence of the SUC2 locus has been placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere based yeast plasmid vector from which invertase is expressed in a Sue" strain of yeast. Oligonucleotide-directed mutagenesis has been used to create a PstI site in the gene at the point encoding the signal peptide cleavage site. An internal methionine codon, the translation start for the cytoplasmic invertase, has been replaced by a serine codon. Mutants in the signal peptide sequence have been produced by replacing the region of the gene upstream of the PstI site with synthetic oligonucleotide cassettes with mixtures of nucleotides at several positions. The mutants could be divided into three classes based on their ability to secrete invertase. The first class of mutants produced secreted invertase, but in reduced amount. There is no obvious correlation between mutation and phenotype. The second class, represented by mutant 4-55B, also exhibited a reduced level of invertase, but a significant fraction (30%) of the enzyme is intracellular. This mutant had a delay in signal peptide cleavage which retards passage of invertase through the secretory pathway. The third class was defective in secretion. Most were defective in translocation from the cytoplasm to the lumen of the endoplasmic reticulum (ER), and produced enzymatically active, non-glycosylated pre-invertase in the cytoplasm. This class of mutant invertases, when transcribed and translated in vitro, was not processed by canine pancreas signal recognition particle (SRP) and microsomes. Comparison of the sequences of the mutant signal peptides of this non-translocating class identifies amino acids at the extreme amino-terminus as the causative defect.<br>Medicine, Faculty of<br>Biochemistry and Molecular Biology, Department of<br>Graduate
36
Cai, Chunhui. "Identification and characterization of yeast synergistic regulatory interaction from high throughput data." HKBU Institutional Repository, 2010. http://repository.hkbu.edu.hk/etd_ra/1190.
Testo completo
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Porter, Susan Dorothy. "Molecular genetic analysis of the saccharomyces cerevisiae Mat Locus." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/29166.
Testo completoAbstract (sommario):
The MAT∝ locus of the yeast Saccharomyces cerevisiae encodes two regulatory proteins responsible for determining the ∝cell type. The MAT∝1 gene encodes ∝1, a positive regulator of ∝cell-specific genes, whereas the MAT∝2 gene encodes a negative regulator of a cell-specific genes (∝2). MAT∝2. (in conjunction with the MATα1 gene) also determines the α/∝ diploid cell type by repressing haploid-specific genes. ∝2 exerts its effect at the transcriptional level in the ∝ cell by binding to a sequence located upstream of α cell-specific genes.
The present study undertook to examine, through in vitro genetic manipulation, the structure/function relationship of the MAT∝ regulatory proteins, particularly∝2, in their role as gene regulators. The construction of mutant MAT∝2 genes containing termination codons at various points within the gene, and subsequent transformation of the mutant genes into mat∝2 yeast, indicated that the carboxy-terminal one-third of the gene product was necessary for full repressor activity in the haploid as well as in the diploid.
A segment within the carboxy-terminal one-third of ∝2 displays some homology to the higher eukaryote homeo domain as well as to a prokaryotic bihelical DNA-binding structural motif. This region of the gene was subjected to semi-random missense mutagenesis in vitro and the mutant genes were analyzed by transformation into strains containing chimaeric genes that encode β-galactosidase from ∝2 and a1/∝2. repressible promoters.
In this manner it was demonstrated that most of those residues in ∝2. which correspond to conserved amino acids in the prokaryotic DNA-binding structure and in the homeo domain are essential for the two repressor activities of ∝2. Several mutations more severely affected the ability of ∝2 to repress α-specific genes than haploid-specific genes.
Analysis of the temperature dependence of the activities of some of the mutants was consistent with the existence of a helix-turn-helix structure at this region of the protein. Finally, further analysis of some of these mutants in vitro confirmed that the observed defect correlated with a loss of DNA-binding activity.<br>Medicine, Faculty of<br>Biochemistry and Molecular Biology, Department of<br>Graduate
38
Herrick, David. "Structural Determinants of mRNA Turnover in Yeast: a Thesis." eScholarship@UMMS, 1989. https://escholarship.umassmed.edu/gsbs_diss/39.
Testo completoAbstract (sommario):
Large differences exist in the decay rates of individual mRNAs yet the molecular basis for such differences is substantially unknown. We have developed a procedure for the measurement of individual mRNAs in the yeast Saccharomyces cerevisiae which utilizes northern or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant strain (RY260). To assess the reliability of half-life measurements obtained in this manner, we have compared the results of this procedure to results obtained by three other procedures (pulse-chase analysis, approach to steady-state labeling, and inhibition of transcription with thiolutin) and also evaluated whether heat-shock alters mRNA decay rates. We find that: i) for most mRNAs, all four procedures yield comparable relative decay rates and ii) there are no significant differences in the mRNA decay rates measured in heat-shocked or non-heat-shocked cells. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MATα1, were unstable (t1/21/2> 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. The following parameters have been analyzed to determine their role in mRNA decay: i) mRNA size; ii) poly(A) tail metabolism; iii) translational status; iv) relative content of rare codons; and v) structures and sequences within the 3'-untranslated region (UTR).
To identify the structural determinants responsible for the rapid decay of the unstable HIS3 and STE2 mRNAs, recombinants of their respective genes were constructed and transformed into strain RY260 on centromere-containing vectors, and the half-lives of the resulting chimeric mRNAs were measured in vivo. Chimeric genes were constructed in which the 3'-UTR of ACT1 was replaced with the corresponding region of the unstable HIS3 or STE2 mRNAs. The decay rate of the ACT1-5'-HIS3-3' mRNA was very similiar to that of the stable endogenous ACT1 mRNA, implying that the 3'-end of HIS3 is not sufficient to transfer the instability phenotype of the HIS3 mRNA. The HIS3-5'-ACT1-3' mRNA from the reciprocal construct was unstable, suggesting that HIS3 instability determinants are located within its 5'-UTR or coding sequence. A 411 nucleotide (nt) deletion within the HIS3 coding region (with either the HIS3 or ACT1 3'-UTR) was stabilized 3-fold suggesting this region is necessary for the rapid decay of HIS3 mRNA. Insertion of these 411 nts in-frame into the entire ACT1 gene had no significant effect on the stability of the hybrid mRNA implying that these HIS3 sequences are not sufficient to function on their own and that they may have to interact with HIS3 5'- sequences. The ACT1-5' -STE2-3' hybrid mRNA decayed with an intermediate half-life of 12 min. Furthermore, an 82% deletion of the STE2 coding region increased the half-life by nearly 2-fold. Both results suggest that instability determinants of STE2 mRNA are not restricted to the 3'-UTR. Our overall conclusion is that mRNA stability is not dictated by simple, transferable elements (sequences or structures), but may involve interactions between multiple determinants in the mRNA.
39
Armstrong, Gareth Owen. "The production of resveratrol by wine yeast." Thesis, Stellenbosch : Stellenbosch University, 2001. http://hdl.handle.net/10019.1/52557.
Testo completoAbstract (sommario):
Thesis (MSc)--Stellenbosch University, 2001.<br>ENGLISH ABSTRACT: Grapevine is constantly under attack from a wide variety of pathogens including viruses,
bacteria and fungi. In order to ensure survival, the grapevine has developed a vast array of
defense mechanisms to combat invading organisms. A key element of this disease
resistance is the production of phytoalexins, of which resveratrol is the primary component.
The synthesis of resveratrol, together with other structural and biochemical defense
mechanisms equips the plant to combat a number of pathogens resulting in the production
of healthy grapes for the vinification of top quality wine. As part of the active disease
response resveratrol is synthesised de novo in the berry skin at the site of infection, on
recognition of the pathogen. Here it is able to limit the damage caused by the pathogen as
well as preventing it from spreading. This gives the plant the opportunity to initiate its
systemic acquired resistance thereby protecting the rest of the plant and preventing
secondary infections.
The fermentation of red wine on the grape skins allows for the extraction of resveratrol
from the skin into the wine. Red wines therefore have a significantly higher concentration
of resveratrol than white varieties, which contain little or no resveratrol at all. It is for this
reason that the moderate consumption of wine, in particular red wine, is synonymous with
a healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol are
important contributors to the cardiovascular benefits derived from the consumption of red
wine. It now seems, however, that significant cardiovascular protection is derived from the
synergistic action of resveratrol, the polyphenols and the alcohol in wine.
With the wholesomeness of any food or beverage being of extreme importance, the
aim of this project was to manipulate wine yeast to produce resveratrol during
fermentation. This required the introduction of an entire metabolic pathway, by integrating
plant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl-
CoA molecules to produce one molecule of resveratrol, Saccharomyces
cerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following genes
were obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanine
ammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4-
hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encoding
CoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-level
expression, the genes were subcloned under the control of the phosphoglycerate kinase
gene (PGK1) promoter and terminator. Due to integration problems with these expression
cassettes and the fact that the yeast was able to consume p-coumaric acid, the 4CL9,
4CL216 and Vst1 (encoding resveratrol synthase) genes were subcloned under the control
of the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids,
respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and
4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produce
resveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformants
expressing the CoA-ligase genes utilised the p-coumaric acid faster than the control,
although it was not possible to determine whether p-coumaroyl-CoA was produced. No
resveratrol was produced under the assay conditions used. The results indicated that the
yeast is unable to produce active resveratrol synthase, which is required to catalyse the
final reaction in the production of resveratrol. Posttranslational modification, such as overglycosylation
and disulphide formation, of the heterologous protein in yeast has been
indicated as the possible reason for the lack of enzyme activity. This introduces an exciting
area of research for the development of biotechnological tools with the ability to increase
the production of active heterologous proteins in yeast.<br>AFRIKAANSE OPSOMMING: Wingerde word voortdurend deur 'n groot verskeidenheid patogene, insluitende virusse,
bakteriee en swamme, aangeval. Ten einde oorlewing te verseker, het die wingerdstok In
wye reeks verdedigingsmeganismes ontwikkel om weerstand te bied teen indringerorganismes.
'n Belangrike faktor in hierdie weerstand teen siektes is die produksie van
fitoaleksiene, waarvan resveratrol die hoofkomponent is. Oeur die sintese van resveratrol,
asook ander strukturele en biochemiese verdedigingsmeganismes, word die plant
toegerus om weerstand te kan bied teen In hele aantal patogene ten einde gesonde
druiwe te produseer wat gebruik kan word vir die vinifikasie van topgehalte wyn. As deel
van die aktiewe reaksie teen siektes, word resveratrol de novo in die dop van die korrel by
die plek van infeksie gesintetiseer sodra 'n patogeen herken word. Hier kan dit die skade
deur die patogeen veroorsaak, beperk en verhoed dat dit versprei. Oit gee aan die plant
die geleentheid om sy sistemies-verworwe weerstand te inisieer, en daardeur die res van
die plant te beskerm, sowel as sekondere infeksies te verhoed.
Die fermentasie van rooiwyn op die druifdoppe maak voorsiening vir die ekstraksie van
resveratrol uit die dop na die wyn. Die konsentrasie van resveratrol in rooiwyn is dus
beduidend hoer as in die wit varietelte, wat geen of baie min resveratrol bevat. Oit is dan
juis die rede waarom die matige inname van wyn, veral rooi wyn, gesien word as In
integrale deel van 'n gesonde leefwyse. Resveratrol se aktiwiteit as antioksidant en antiinflammatoriese
middel lewer In belangrike bydrae tot die kardiovaskulere voordele wat
verkry word uit die inname van rooiwyn. Oit blyk egter nou dat die beduidende
kardiovaskulere beskerming gesetel is in die sinergistiese werking van resve ratro I, die
polifenole en die alkohol in wyn.
Aangesien die heilsaamheid van enige voedsel of drank van die uiterste belang is,
was dit die doel van hierdie projek om wyngis te manipuleer ten einde tydens die
fermentasieproses resveratrol te produseer. Hiervoor moes 'n volledige metaboliese pad
daargestel word deur plantgene in die gis te inkorporeer. Resveratrol-sintase maak
gebruik van drie maloniel-KoA-molekules en een p-kumarotel-Kos-molekule om een
molekule resveratrol te produseer. Saccharomyces cerevisiae produseer maloniel-KoA,
maar nie p-kumaroiel-Kcs, nie. Oie volgende gene is dus aangewend om die gis in staat
te stel om p-kumarolel-Koe, te produseer: PAL, wat fenielalanien-ammoniak-liase
enkodeer om fenielalanien om te sit na kaneelsuur; C4H, wat sinnamaat-4-hidroksliase
enkodeer om kaneelsuur om te sit na p-kumaarsuur; en 4CL9 of 4CL216 wat KoA-ligases
enkodeer om p-kumaarsuur om te sit na p-kumarolel-Kos, Om hoevlak-uitdrukking te
verkry, is die gene gesubkloneer onder beheer van die fosfogliseraat-kinase-geen(PGK1)-
promotor en -terminator. As gevolg van integrasieprobleme met hierdie
uitdrukkingskassette en die feit dat die gis die p-kumaarsuur kon verteer, is die 4CL9-,
4CL216- en Vst1- (wat resveratrol-sintase enkodeer) gene na episomale plasmiede
gesubkloneer onder beheer van die alkohol-dehidrogenase(ADH2)- en PGK1-promotors onderskeidelik. 'n Laboratorium-gisstam wat 6f beide die Vst1-geen en die 4CL9-geen, 6f
die Vst1-geen en die 4CL216-geen bevat het, is geevalueer vir die verrnoe om pkumaarsuur
te benut en resveratrol te produseer. Noordelike klad analises het bevestig
dat die Vst1-, 4CL9- en 4CL216-gene getranskribeer en ooruitgedruk was in vergelyking
met die kontrole-stam. Die transformante wat die KoA-ligases uitgedruk het, het die pkumaarsuur
vinniger benut as wat die kontrole dit gedoen het, alhoewel dit nie moontlik
was om vas te stel of o-kurnarotel-Kos, geproduseer is nie. Met die essai-kondisies wat
gebruik is, is geen resveratroI geproduseer nie. Die resultate het daarop gedui dat die gis
nie daartoe in staat is om aktiewe resveratrol-sintase, wat nodig is vir die katalise van die
finale reaksie in die produksie van resveratrol, te produseer nie. Naomsettingsmodifikasies
van die heteroloe protelen in die gis, soos oor-glikosilasie en
disulfiedvorming, is aangewys as die moontlike rede vir die gebrek aan ensiemaktiwiteit.
Dit stel In opwindende veld vir verdere navorsing voor, naamlik die ontwikkeling van
biotegnologiese middele met die vermoe om die produksie van aktiewe heteroloe
protelene in gis te verhoog.
40
Ruz-Peres, Monica. "Avaliação da presença de fungos em amostras de leite cru e estudo da susceptibilidade destes microrganismos às relações temperatura/tempo empregadas nos processos de pasteurização e fervura." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-18062008-140110/.
Testo completoAbstract (sommario):
O presente trabalho foi delineado considerando-se a importância de fungos filamentosos e leveduras, os quais estão associados a diversas patologias no homem e animais. Deve-se considerar ainda que o leite e seus derivados lácteos contaminados com estes microrganismos podem constituir potenciais vias de transmissão de zoonoses a eles relacionados. Foram analisadas amostras de leite cru dos tipos A, B e C colhidas nas próprias propriedades leiteiras, bem como amostras de leite comercializado diretamente ao consumidor, visando-se a comparação da qualidade destes produtos quanto à presença e quantidade de fungos. Procedeu-se ainda à avaliação da susceptibilidade dos fungos isolados das amostras de leite às relações temperatura/tempo empregados nos processos de pasteurização e avaliação da sensibilidade destes isolados à fervura do leite. Foram analisadas 70 amostras de leite, sendo 50 de tanques de refrigeração de propriedades leiteiras, 10 de latões de propriedades de exploração leiteira e 10 de latões de transportadores e distribuidores que armazenam e comercializam leite clandestino, sendo que não houve diferença estatisticamente significante entre as medianas das quantidades de unidades formadoras de colônias de fungos/mL de Leite das diferentes procedências, indicando que o nível de contaminação por fungos nas amostras destas três origens foi similar. Foram isolados, em diferentes percentagens, fungos filamentosos e leveduras de todas as amostras de leite das diferentes procedências: Candida spp. (C. krusei, C. guilliermondii, C. tropicalis, C. kefyr, dentre outras), Geotrichum spp., Rhodotorula spp., Trichosporon spp., Aureobasidium sp., Penicillium spp., Acremonium spp., Chrysosporium sp., Mucor spp., Aspergillus spp. Todos os gêneros e espécies isoladas foram submetidos aos testes de pasteurização rápida e lenta, bem como a fervura. O processo de pasteurização rápida foi o procedimento no qual houve maior resistência (72,18%) por parte dos isolados de leveduras e fungos filamentosos submetidos ao teste na metodologia aplicada, seguido pelo processo de fervura (15,89%) e o de pasteurização lenta (0,99%).<br>The present study was outlined considering the importance of filamentous fungi and yeasts, associated to various pathologies in man and animals. It must be considered that milk and dairy products when contaminated with these microorganisms can represent a potential means of zoonosis transmission related to them. Samples of raw milk types A, B and C, collected in dairy farms, as well as milk sold directly to the consumer, were analysed, aiming the comparison of the quality of these products as to the presence and quantity of fungi. The susceptibility of the isolates to the temperature/time rates employed in pasteurization and boiling was also evaluated. Seventy samples of raw milk were analysed, 50 from bulk tanks, 10 from small tanks of milk from dairy farms, and 10 from transportation tanks and distributors that store and sell clandestine milk. No statistically significant difference was observed between medians of the quantity of colonies forming units of fungi/mL of milk from the different sources, indicating that the level of contamination by fungi was similar when the three sources were compared. Filamentous fungi and yeasts were isolated in different percentages from all the samples from the different sources: Candida spp. (C. krusei, C. guilliermondii, C. tropicalis, C. kefyr, etc.), Geotrichum spp., Rhodotorula spp., Trichosporon spp., Aureobasidium sp., Penicillium spp., Acremonium spp., Chrysosporium sp., Mucor spp., Aspergillus spp. All isolates were submitted to pasteurization and boiling. Quick pasteurization was the procedure in which the highest resistance (72.18%) of the isolates was verified, followed by boiling (15.89%) and slow pasteurization (0.99%).
41
Lau, Choi-yi Candy, and 劉彩怡. "Characterization and application of MP1 homologues in penicillium marneffei." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B42841203.
Testo completo
42
Tekolo, Obakeng McDonald. "The osmotic stress response of basidiomycetous yeasts." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/18703.
Testo completoAbstract (sommario):
Thesis (MSc)--University of Stellenbosch, 2007.<br>ENGLISH ABSTRACT: Basidiomycetous yeasts are found in a wide range of geographical areas ranging from tropical forests to desert regions. These yeasts are associated with different habitats such as soil, decaying vegetative debris, living plants and animals. Some may even be opportunistic human pathogens. In most of these habitats the yeasts may periodically be exposed to adverse conditions such as osmotic stress. Forty-one basidiomycetous yeast strains obtained from culture collections and isolated from nature were studied using various methods which includes the determination of different minimum water activities (aw; NaCl or sorbitol) for growth, survival in soil of varying moisture content, intracellular osmolytes accumulated and their release upon hypo-osmotic stress. The growth of most strains showed greater tolerance to NaCl than sorbitol at the same level of water activity. Interestingly, there were no basidiomycetous strains that showed growth below 0.90aw.
13C nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC) was used to analyze the osmolytes accumulated by all the strains of basidiomycetous yeasts when grown at 0.95 aw (NaCl). Glycerol was the major solute accumulated intracellulary by all the yeasts. Arabitol, mannitol or trehalose was accumulated in addition to glycerol in most yeasts whereas a number of yeasts only accumulated glycerol when grown at reduced aw. However, Cryptococcus laurentii US 1F was an exception by accumulating three solutes intracellularly when grown at reduced water activity. When exposed to hypo-osmotic shock all three solutes were rapidly released from the cells. Cryptococcus hungaricus CBS 5421, Cryptococcus macerans CBS 2206 and Cryptococcus neoformans US I1 were further analyzed with 13C NMR spectroscopy to determine whether the type of osmolyte accumulated during different phases of growth at 0.95aw (NaCl) might change. No changes were observed as the same osmolytes were accumulated in all cases.
Five yeast strains (C. neoformans US I1, Rhodotorula mucillaginosa CBS 5951, C. macerans CBS 2206T, Filobasidium floriforme CBS 6240 and Sporidiobolus salmonicolor CBS 5937) were analyzed by HPLC for osmolytes released when exposed to hypo-osmotic shock. The strains differed in the pattern of response of osmolyte release. Only three strains released most of their osmolytes rapidly within 5 min, while C. macerans CBS 2206T and R. muculaginosa CBS 5951 retained most of the osmolytes intacellularly. This suggests that there might be different mechanisms of osmolyte release in basidiomycetous yeasts.
A few strains of basidiomycetous yeasts (C. neoformans US I1, R. mucilaginosa CBS 5951, C. laurentii 1F, C. macerans CBS 2206T, F. floriforme CBS 6240, C. neoformans CBS 0132, C. laurentii CBS 0139, S. salmonicolor CBS 5937 and Filobasidium capsuligenum CBS 4381) were grown in soil cultures of different field capacity (100%, 25%, 10% and 5%) and evaluated for their survival in this environment. All the strains grew at 100% field capacity. Strains R. mucilaginosa CBS 5951, F. floriforme CBS 6240 and F. capsuligeum CBS 4381 also showed growth in soil at 25% field capacity. However, strains C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and C. laurentii CBS 0139 did not grow at this moisture content but survived up until the end of the experimental period. At lower soil moisture content (5% and 10% field capacity), the yeast strains either showed survival or decreased viability towards the end of the experimental period. Strain C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and R. mucilaginosa CBS 5951 and F. floriforme CBS 6240 showed survival at both 5% and 10% field capacity. However, strain F. neoformans CBS 0132, C. laurentii CBS 0139, F. capsuligenum CBS 4381 and S. salmonicolar CBS 5937 showed a decrease in viability after either 2 or 5 days of incubation. No relationship could be found between the type and number of intracellular osmolytes accumulated when exposed to osmotic stress (0.95aw NaCl) and the ability to grow and survive in soil with lower moisture content. Similarly, the ability of the yeasts to grow and survive in soil with lower moisture content did not correlate with their minimum aw for growth in a liquid medium. It was speculated that other factors, such as the physico-chemical composition of the soil, may also play a role in the survival of a particular yeast species in soil. This study has shown that the responses of basidiomycetous yeasts to reduced aw are physiologically similar to the ascomycetous yeasts. The types of osmolytes accumulated are similar but the basidiomycetous yeasts appear to be more sensitive to reduced aw and they tolerate NaCl better than sorbitol whereas the ascomycetous yeasts tolerate high sugar environments better. This is in agreement with the environments where these yeasts are usually found.<br>AFRIKAANSE OPSOMMING: Basidiomisete giste word aangetref in ‘n wye reeks geografiese areas, wat strek vanaf tropiese woude tot woestynstreke. Hierdie giste word geassosieer met verskillende habitatte soos grond, verrottende vegetatiewe reste, lewende plante en diere. Sommige mag selfs opportunistiese menslike patogene wees. By meeste van hierdie habitatte mag giste periodies blootgestel word aan moeilike toestande soos osmotiese stres. Een-en-veertig basidiomisete gisrasse, verkry vanaf kultuurversamelings en geisoleer vanuit die natuur, was bestudeer met verskeie metodes, waaronder die bepaling van verskillende minimum water aktiwiteite (aw; NaCl of sorbitol) vir groei, droë massa bepalings, akkumulasie van intrasellulêre osmoliete, asook hul vrystelling met hipo-osmotiese stres. Meeste rasse het meer weerstand teen NaCl as sorbitol gehad by dieselfde vlak van wateraktiwiteit. Dit was interessant om op te let dat geen basidiomisete stamme groei onder 0.90aw getoon het nie.
Beide 13C kern magnetiese resonansie (KMR) spektroskopie en hoë uitset vloeistof chromatografie (HUVC) was gebruik om alle opgehoopde osmoliete te analiseer vir alle basidiomisete gisrasse tydens groei tot by 0.95 aw (NaCl). Vir alle giste was gliserol die opgelosde stof wat die meeste intrasellulêr opgehoop het. Arabitol of mannitol of trehalose het saam met gliserol in meeste giste opgehoop, terwyl ‘n aantal giste slegs gliserol opgehoop het tydens groei by verlaagde aw. Cryptococcus laurentii US 1F was daarenteen ‘n uitsondering deurdat dit drie opgelosde stowwe intrasellulêr versamel het tydens groei by verlaagde wateraktiwiteit. Al drie hierdie opgelosde stowwe is uit die selle vrygesel na blootstelling aan hipo-osmotiese skok. Verdere 13C KMR spektroskopie analise was gedoen op Cryptococcus hungaricus CBS 5421, Cryptococcus macerans CBS 2206 en Cryptococcus neoformans US I1 om vas te stel of die tipe opgehoopde osmoliet tydens verskillende fases van groei by 0.95aw (NaCl) mag verander. Geen veranderinge was egter waargeneem aangesien dieselfde osmoliete in alle gevalle opgehoop het.
Vyf gisrasse (C. neoformans US I1, Rhodotorula mucillaginosa CBS 5951, C. macerans CBS 2206T, Filobasidium floriforme CBS 6240 en Sporidiobolus salmonicolor CBS 5937) was geanaliseer deur HUVC vir osmolietvrystelling tydens blootstelling aan hipo-osmotiese skok. Die rasse het verskil in hul responspatroon van osmolietvrystelling. Slegs drie rasse het meeste van hul osmoliete vrygestel binne die eerste 5 minute, terwyl ander, C. macerans CBS 2206T en R. muculaginosa CBS 5951 meeste van hul osmoliete intrasellulêr teruggehou het. Dit dui daarop dat daar dalk verskillende meganismes van osmolietvrystelling in basidiomisete giste mag voorkom.
Etlike basidiomisete gisrasse (C. neoformans US I1, R. mucilaginosa CBS 5951, C. laurentii 1F, C. macerans CBS 2206T, F floriforme CBS 6240, C. neoformans CBS 0132, C. laurentii CBS 0139, (B) S. salmonicolor CBS 5937, Filobasidium capsuligenum CBS 4381) was opgegroei in grondkulture van verskillende veldkapasiteit (100%, 25%, 10% en 5%) en ondersoek vir hul oorlewing in hierdie omgewing. Al die rasse kon groei by ‘n 100% veldkapasiteit. Die rasse R. mucilaginosa CBS 5951, F. floriforme CBS 6240 en F. capsuligeum CBS 4381 kon ook groei in grond met 25% veldkapasiteit. Alhoewel rasse C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T en C laurentii CBS 0139 nie kon groei by hierdie voginhoud nie, het hulle nog steeds oorleef tot aan die einde van die eksperimentele tydperk. By verlaagde grond voginhoud (5% en 10% veldkapasiteit) het die gisrasse of oorleef of ‘n verlaagde lewensvatbaarheid openbaar teen die einde van die eksperimentele prosedure. Die rasse C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and R. mucilaginosa CBS 5951 en F. floriforme CBS 6240 het oorleef by beide 5% en 10% veldkapasiteit. Die rasse F. neoformans CBS 0132, C. laurentii CBS 0139, F. capsuligenum CBS 4381 en S. salmonicolar CBS 5937 het egter ‘n verlaging in lewensvatbaarheid getoon na ‘n periode van of 2 of 5 dae inkubasie. Geen ooreenstemming kon gevind word tussen die tipe en aantal intrasellulêre opgehoopde osmoliete nie na blootstelling aan osmotiese druk (0.95aw NaCl), asook hul vermoë om te groei en oorleef in grond met ‘n laer voginhoud. Terselfdertyd het die giste se vermoë om te groei en oorleef in grond met ‘n laer voginhoud nie ooreengestem met hul minimum aw vir groei in vloeibare groeimedium nie. Ter spekulasie kan ander faktore, soos die fisio-chemiese samestelling van die grond ook ‘n rol speel in die oorlewing van ‘n spesifieke gisspesie in grond.
Hierdie studie het getoon dat basidiomisete giste se respons tot verlaagde aw fisiologies dieselfde is as vir askomisete giste. Alhoewel die tipe versamelde osmoliete identies was, wil dit voorkom asof basidiomisete giste meer sensitief teenoor ‘n verlaagde aw is, tesame met ‘n beter bestandheid teen NaCl as sorbitol, terwyl askomisete giste hoë suiker omgewings beter kan verdra. Dit is in ooreenstemming met die tipe omgewings waar hierdie giste gewoonlik voorkom.
43
Kriel, Johan Hendrik. "Development of synthetic signal sequences for heterologous protein secretion from Saccharomyces cerevisiae." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53364.
Testo completoAbstract (sommario):
Thesis (MSc)--Stellenbosch University, 2003.<br>ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and
involve the interplay of a multitude of proteins. A unique collection of thermosensitive
secretory mutants allowed scientists to demonstrate that the secretory pathway of the
yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All
proteins commence their journey in the endoplasmic reticulum, where they undergo
amino-linked core glycosyl modification. After passage through the Golgi apparatus,
where the remodelling of the glycosyl chains is completed, proteins are transported to
their final destinations, which are either the cell surface, periplasmic space or the
vacuole.
Proteins destined for secretion are usually synthesised with a transient
amino-terminal secretion leader of varying length and hydrophobicity, which plays a
crucial role in the targeting and translocation of their protein cargo. Considerable
effort has been made to elucidate the molecular mechanisms involved in these
processes, especially due to their relevance in a rapidly expanding biotech industry.
The advantages of S. cerevisiae as a host for the expression of recombinant
proteins are well documented. Unfortunately, S. cerevisiae is also subject to a
number of drawbacks, with a relative low product yield being one of the major
disadvantages.
Bearing this in mind, different secretion leaders were compared with the aim of
improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the
S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for
its ability to degrade raw starch and an improved secretion of its amylase enzymes
from S. cerevisiae paves the way for a potential one-step starch utilisation process.
Three sets of constructs were prepared containing the LKA 1 and LKA2 genes
separately under secretory direction of either their native secretion leader, the
S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1
secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The
inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p
proteolytic processing of the leader/protein fusion. Strains expressing the amylase
genes under their native secretion leaders resulted in the highest saccharolytic
activity in the culture medium. In contrast to this, strains utilising the synthetic
secretion leader produced the highest fermentation yield, but had a lower than
expected extracellular activity. We hypothesise that the native amylase leaders may
function as intramolecular chaperones in the folding and processing of their
passenger proteins, thereby increasing processing efficiency and concomitant
enzyme activity.<br>AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en
betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke
versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat
gestelom die ooreenkoms tussen die sekresiepad van die gis
Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle
proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook
amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur
die Golgi-apparaat beweeg het, waar die laaste veranderings aan die
glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die
seloppervlak, die periplasmiese ruimte of die vakuool, vervoer.
Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike,
amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul
proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend
om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as
gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf.
Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante
proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die
relatiewe lae produkopbrengs die belangrikste is.
Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met
die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die
S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is
bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy
amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap
styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene
onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die
S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n
sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van
'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p
proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene
onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese
aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die
sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar
met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse
amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul
proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.
44
Zhang, Daiyuan Pirtle Robert M. "Expression analysis of the fatty acid desaturase 2-4 and 2-3 genes from Gossypium hirsutum in transformed yeast cells and transgenic Arabidopsis plants." [Denton, Tex.] : University of North Texas, 2008. http://digital.library.unt.edu/permalink/meta-dc-9002.
Testo completo
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Lau, Choi-yi Candy. "Characterization and application of MP1 homologues in penicillium marneffei." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B42841203.
Testo completo
46
Wang, You. "Development of yeast-based methods to screen for plant cytokinin-binding proteins." Access electronically, 2004. http://www.library.uow.edu.au/adt-NWU/public/adt-NWU20060123.141512/index.html.
Testo completo
47
Flanagan, Joan Frances. "The Yeast SWI/SNF Complex Structure and Function: A Dissertation." eScholarship@UMMS, 2001. https://escholarship.umassmed.edu/gsbs_diss/187.
Testo completoAbstract (sommario):
DNA is packaged within the cells' nucleus as a highly compact chromatin structure ranging between 100-400 nm fibers. The organization and alteration of this structure is mandatory in order to arbitrate DNA-mediated processes of the cell, including transcription, DNA replication, recombination and repair. Many different kinds of enzymes modify chromatin components and, in turn, regulate the accessibility of DNA. These multi-subunited enzymes have emerged as key regulators for several processes of the cell. Central to understanding how DNA-mediated processes are regulated is to comprehend the consequences of these modifications of chromatin, which lead to altered states of either activation or inactivation.
One class of factors known to modify chromatin structure is the ATP-dependent chromatin remodeling enzymes. This class of enzymes encompasses evolutionarily conserved multi-subunited enzymes, which appear to function by using the energy of ATP hydrolysis to disrupt histone-DNA interactions. The prototype of ATP-dependent chromatin remodelers is the Saccharomyces cerevisiae SWI/SNF complex. The yeast SWI/SNF complex is required for the full functioning of several transcriptional activators and for the expression of a subset of yeast genes, a notable number being inducible and mitotic genes. The purified complex is comprised of the following eleven different polypeptides: Swi2p/Snf2p, Swi1p, Swi3p, Snf5p, Snf6p, Swp73p, Arp7p, Arp9p, Swp82p, Swp29p and Snf11 p. It has been established that a core of homologous subunits (Swi2p, Swi3p, Swp73p, Snf5p and the Arp proteins) is conserved among the SWI/SNF-related complexes from several organisms (yRSC, hSWI/SNF, hRSC, DrosophilaBrahma). However, the functional contribution of these polypeptides in the complexes for altering chromatin structure is largely unknown. In this study, biochemistry is used to examine the structure of the complex and function of individual subunits of the yeast SWI/SNF complex to understand better how these proteins are acting in concert to remodel chromatin. In addition, we examine a role for SWI/SNF complex in the process of DNA replication.
The relative stoichiometry of the SWI/SNF complex subunits was determined by in vitrobiochemical studies. Co-immunoprecipitation has demonstrated that there is only one copy of Swi2p/Snf2p per complex. Subsequent radioactive labeling of the purified complex revealed that the complex contains one copy of each subunit per complex with the exception of Swi3p and Snf5p, which are present in two copies per complex.
The subunit organization of SWI/SNF complex has been more clearly defined by determining direct subunit-subunit interactions in the complex. The Swi3p component has previously been shown to be critical for complex function in vivo and essential for the integrity of the complex in vitro, and this study demonstrates that Swi3p serves as a scaffolding protein that nucleates SWI/SNF complex assembly. In vitrobinding studies with Swi3p have revealed that Swi3p displays self-association, as well as direct interactions with the Swi2p, Snf5p, Swp73p, Swi1p and Snf6p members of the complex.
The direct interactions of the yeast SWI/SNF subunits with transcriptional activators, thought to be important for yeast SWI/SNF targeting, were examined. In vitrobinding assays demonstrate that individual SWI/SNF subunits, Snf5p, Snf6p and Swi1p, and sub-complexes Swi2p/Swi3p and Swp73p/Swi3p can directly interact with specific domains of transcriptional activators of either the Swi5p zinc-finger DBD or VP16 acidic activation domain. This work begins to characterize the functional contribution of individual subunits, and cooperative sub-complexes that are critical for the SWI/SNF complex functional activities.
The yeast SWI/SNF complex was investigated for the ability to playa role in DNA replication. Interestingly, plasmid stability assays reveal that minichromosomes that contain DNA replication origin ARS121 is weakened when the SWIISNF complex is non-functional. ARS121's SWI/SNF dependency is overcome by the over-expression of DNA replication regulatory protein, Cdc6p. Thus, this suggests SWI/SNF may either indirectly effect DNA replication by effecting the expression of Cdc6p, or has a redundant function with Cdc6p. In addition, several crippled derivatives of ARS1 acquire SWI/SNF dependence, and it is found that the SWI/SNF complex requires a transcriptional activation domain to enhance ARS1 function. These results reinforce the view that SWI/SNF play a role in two chromatin-mediated processes', transcription and DNA replication.
48
Yan, Zhun Xu Jianping. "Mating system and mitochondrial inheritance in a basidiomycete yeast, Cryptococcus neoformans." *McMaster only, 2006.
Cerca il testo completo
49
Guerreiro, Marco Alexandre Figueira Rodrigues. "Unveiling the mating system and genetic variability in the yeast Kwoniella mangroviensis using molecular approaches." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/8288.
Testo completoAbstract (sommario):
Dissertação para obtenção do Grau de Mestre em
Genética Molecular e Biomedicina<br>In fungi, sexual reproduction is orchestrated by genomic regions known as mating type loci (MAT), which can be defined by two physically unlinked sex determining regions (tetrapolar mating system) or a single locus (bipolar system). Kwoniella mangroviensis is a saprobic basidiomycetous yeast, belonging to the Kwoniella clade of the order Tremellales, which was described as possessing a bipolar mating-system, similar to the related pathogenic species Cryptococcus neoformans and Cryptococcus gattii of the sister Filobasidiella clade. Studies aimed at elucidating the evolution of the MAT locus of these two Cryptococcus species of clinical importance, targeted several related saprobic species in the Kwoniella and Filobasidiella clades. An evolutionary model ensuing from those studies suggests that the tetrapolar mating-systems found in most species are ancestral and gave rise to the bipolar mating-system of C. neoformans by chromosomal rearrangements and fusion events.
The present study comprised strains from the original work describing K. mangroviensis, as well as additional isolates from plant substrates in Europe and Africa. A multilocus sequence typing approach revealed genetic variability among those strains and led to the identification of two novel species closely related to K. mangroviensis: Kwoniella sp. A and Kwoniella sp. B. The mating system of K. mangroviensis and sibling species was further explored by a genetic approach based on sequencing of two MAT genes: STE20 and the divergently transcribed genes SXI1 and SXI2. The results obtained demonstrated tetrapolar mating systems in K. mangroviensis, as well as in Kwoniella sp. A and Kwoniella sp. B. Additionally, the MAT locus structure of K. mangroviensis was unveiled by sequencing a 43 kb genomic region containing the STE20 gene. Twelve genes also present in the MAT loci of related species were identified, and full synteny was found between K. mangroviensis and Cryptococcus heveanensis, a distant member of the Kwoniella clade. These findings provided novel insights into the evolution of MAT loci in basidiomycetous yeasts in the Tremellales.<br>Fundação para a Ciência e Tecnologia - PTDC/BIA-MIC/113051/2009
50
Smit, Annel. "Engineering yeast for the production of optimal levels of volatile phenols in wine." Thesis, Stellenbosch : Stellenbosch University, 2002. http://hdl.handle.net/10019.1/52823.
Testo completoAbstract (sommario):
Thesis (MSc)--University of Stellenbosch, 2002.<br>ENGLISH ABSTRACT: Phenolic acids (principally p-coumaric and ferulic acids), which are generally esterified with
tartaric acid, are natural constituents of grape must and wine, and can be released as free
acids during the winemaking process by certain cinnamoyl esterase activities. Free phenolic
acids can be metabolised into 4-vinyl and 4-ethyl derivatives by several microorganisms
present in wine. These volatile phenols contribute to the aroma of the wine.
The Bretfanomyces yeasts are well known for their ability to form volatile phenols in
wine. However, these species are associated with the more unpleasant and odorous
formation of the ethylphenols and the formation of high concentrations of volatile phenols.
Other organisms, including some bacterial species, are responsible for the formation of
volatile phenols at low concentrations, especially the 4-vinylphenols, and this enhances the
organoleptic properties of the wine.
The enzymes responsible for the decarboxylation of phenolic acids are called phenolic
acid decarboxylases; and several bacteria and fungi have been found to contain the genes
encoding these enzymes. The following genes have been characterised: PAD1 from
Saccharomyces cerevisiae, fdc from Bacillus pumilus, pdc from Lactobacillus plantarum and
padc from Bacillus subtilis. PadA from Pediococcus pentosaceus was also identified.
S. cerevisiae contains the PAD1 (phenyl acrylic acid decarboxylase) gene, which is
steadily transcribed in yeast. The activity of the PAD1-encoded enzyme is low. Phenolic
acid decarboxylase from B. subtilis, as well as p-coumaric acid decarboxylase from
L. plantarum displays substrate inducible decarboxylating activity with phenolic acids. Both
the p-coumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) genes
were cloned into PGK1 PT expression cassette. The PGK1 PT expression cassette consisted
of the promoter (PGK1 p) and terminator (PGK1 T) sequence of the yeast
phosphoglyceratekinase I gene (PGK1). Episomal and yeast integration plasmids were
constructed for the PAD1 gene under the control of the PGK1 PT for overexpresion in yeast.
Industrial strains with the PAD1 gene disrupted were also made. Overexpression of pcoumaric
acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) in S. cerevisiae
showed high enzyme activity in laboratory strains. The overexpressed PAD1 gene did not
show any higher enzyme activity than the control strain. Both bacterial genes, under the
control of the PGK1 PT cassette, were also cloned into a yeast-integrating plasmid, with the
SMR1 gene as selective marker. The cloning and transformation of pdc and padc into
industrial wine yeast strains can therefore be used to detect the effect of phenolic acid
decarboxylase genes in the winemaking process for the possible improvement of wine
aroma. Wine was made with all three strains (the bacterial genes overexpressed and PAD1
disrupted). The effect of these genes in wine was determined through GC analysis. The results showed that the bacterial genes could effectively produce higher levels of volatile
phenols in the wine. The manipulated strains also produced enzymes capable of producing
large amounts of favourable monoterpenes in the wine.
This study paves the way for the development of wine yeast starter culture strains for
the production of optimal levels of volatile phenols, thereby improving the sensorial quality of
wine.<br>AFRIKAANSE OPSOMMING: Die fenoliese sure (p-kumaarsuur en ferolsuur), wat as natuurlike komponente in mos en wyn
voorkom, word gewoonlik as esterverbindings in wynsteensuur gevind. Seker
esterase-aktiwiteite kan die fenoliese sure as vrye sure vrystel gedurende die
wynmaakproses. Hierdie vrye fenoliese sure kan dan weer deur verskillende mikroorganismes
na 4-viniel en 4-etiel derivate omgesit word. Hierdie derivate staan as vlugtige
fenole bekend en kan tot die aroma van wyn bydra.
Die Brettanomyces giste is baie bekend vir hulle vermoeë om vlugtige fenole in wyn te
vorm, maar dit is gewoonlik die formasie van hoë konsentrasies van vlugtige fenole, veral die
4-etiel derivate, wat met af geure geassosieer word. Ander organismes besit egter die
vermoeë om vlugtige fenole teen lae konsentrasies te vorm, veral die 4-viniel derivate, wat 'n
aanvullende effek op die wyn aroma kan hê. .
Die ensieme wat verantwoordelik is vir die dekarboksilasie van fenoliese sure staan as
fenolsuurdekarboksilases bekend. Verskeie bakterieë en fungi bevat gene wat vir hiedie
ensieme kodeer. Die volgende gene is reeds gekarakteriseer: PAD1 van
Saccharomyces cerevisiae, fdc van Bacillus pumilus, pdc van Lactobacillus plantarum en
padc van Bacillus subtilis. PadA van Pediococcus pentosaceus is ook reeds geïdentifiseer.
S. cerevisiae bevat die PAD1- (fenielakrielsuurdekarboksilase) geen, wat teen 'n vaste
tempo in gis getranskribeer word. Die aktiwiteit van hierdie ensiem is egter laag.
Fenolsuurdekarboksilase van B. subtilis, sowel as p-kumaarsuurdekarboksilase van
L. plantarum, vertoon "n substraat-induseerbare dekarboksilerende aktiwiteit met fenoliese
sure. Beide die p-kumaarsuur dekarboksilase en die fenolsuurdekarboksilase gene is in die
PGK1PT ekspressie kasset gekloneer. Episomale en gisintegreringsplasmiede is vir die
PAD1-geen onder beheer van die PGK1 PT ekspressiekasset gekonstrueer vir die
ooruitdrukking van hierdie geen in gis. Die PGK1 PT ekspressiekasset het bestaan uit die
promotor- (PGK1 p) en termineerdersekwense (PGK1 T) van die gisfosfogliseraatkinasegeen
(PGK1). Industriële gisrasse is ontwikkel waarin die PAD1-geen onderbreek is.
Ooruitdrukking van p-kumaarsuurdekarboksilase (Pdc) en fenolsuurdekarboksilase (pade) in
S. cerevisiae toon hoë ensiemaktiwiteit in laboratoriumgisrasse. Die ooruitdrukking van die
PAD1-geen het nie hoër aktiwiteit as die kontroleras gewys nie. Albei die bakteriële gene,
onder die beheer van die PGK1 PT ekspressiekasset, is ook in "n gisintegreringsplasmied met
die SMR1-geen as selektiewe merker geplaas. Die klonering en transformasie van pdc en
padc in industriële wyngiste kan dus gebruik word vir die bepaling van die effek van fenolsuur
dekarboksilases in die wynmaakproses en die moontlike verbetering van wynaroma. Wyn is
met al drie die industriële rasse (die ooruitgedrukte bakteriële gene en die ontwrigte PAD1- geen) gemaak. Die effek van die teenwoordigheid van hierdie gene in die wynmaakproses is
deur gaschromatografie bepaal. Die resultate het aangedui dat die bakteriële gene op In
effektiewe wyse vlugtige fenole in die wyn kan produseer. Sekere monoterpene is ook in In
verhoogde mate gedurende hierdie proses gevorm.
Hierdie studie baan die weg vir die ontwikkeling van reingisinentingskulture vir die
produksie van optimale vlakke van vlugtige fenole om sodoende die sensoriese gehalte van
die wyn te verbeter.