Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2018 / Beer is one of the most widely consumed alcoholic beverages in the world. The brewing process is based on natural enzymatic activities that take place during the malting of barley grain, mashing of grist and fermentation of wort. Insufficient malt enzyme activity during the mashing process leads to high levels of barley β-glucan, arabinoxylan (AX) and dextrins in the wort as well as in the final beer. It was reported that high levels of β-glucan and AX increase wort and beer viscosity which lower the rate of beer filtration and this negatively affect the production rate in the brewery. During beer fermentation, brewing yeast catalyses the conversion of wort sugars to ethanol, carbon dioxide and other metabolic products. However, non-fermentable carbohydrates i.e., limit dextrins remain in the wort and final beer. These non-fermentable carbohydrates are known to contribute to the caloric value of beer which might lead to weight gain in consumers. The objectives of this study were to evaluate the effect of recombinant yeast strains expressing an endo-β-1,4-glucanase or an endo-β-1,4-xylanase on beer viscosity (as an indicator of filterability) and an α-amylase on residual sugars levels. The effect of the above mentioned enzymes on the aroma, appearance, flavour, mouth-feel and overall quality of the beer was also determined. Wort was produced in the University of Limpopo micro-brewery and the wort was pitched with different recombinant strains. The wild-type strain served as control. The results obtained showed that the xylanase expressing strain produced a measurable decrease in viscosity over the course of the fermentation, but endo-glucanase did not have any effect on the beer viscosity. The α-amylase producing strain, did not show a measurable reduction of residual sugars in the final beer probably as a result of very low activity on α-1,6 glycosidic bonds in dextrins during fermentation. The xylanase and α-amylase producing strain fermented effectively with good attenuation (decrease in wort specific gravity). The beer produced by the α-amylase and control strains were preferred in terms of taste and had similar qualities. The secreted amylolytic activity was not sufficient to significantly reduce residual sugar in the final beer. Although the xylanase secreting strain produced a beer with lower viscosity, the enzyme had a negative impact on the taste of the beer. Key words: Brewer’s yeast, beer fermentation, low calorie beer, amylase, xylanase, endo-glucanase.

Among the various external signals perceived by yeast cells, nutrient availability is a condition to which these cells show a highly diverse biological response. Diploid cells in response to different nutritional stress conditions shows different developmental outcomes. On nitrogen starvation, cells undergo dimorphic transition whereby a unicellular yeast form transforms to a multicellular pseudohyphal form. While in the complete absence of a nitrogen source and a fermentable carbon source, yeast cells enter into a complex developmental program termed sporulation which culminates in haploid spores. The main objective of this work was to understand the role played by S. cerevisiaeSUB1 in starvation-induced meiotic program of diploid cells, decipher its target in sporulation specific gene expression cascade, study the domain architecture of Sub1 and examine its functional homology to mammalian PC4. Role of Sub1 in induction of sporulation and other stress responses in S. cerevisiae In a previous whole-genome screen for mutants with altered sporulation efficiency in the Saccharomyces cerevisiae S288c strain, SUB1 locus was identified as a negative regulator of sporulation (Deutschbaueret al., 2002). Moreover, genome-wide gene expression analysis in SK1 strain had shown that SUB1 transcript levels are repressed during sporulation (Chu et al., 1998). Many studies in different yeast strain backgrounds implicate more than 1,000 genesout of 6,200 genes in yeast genome as being differentially expressed during the sporulation process (Chu et al., 1998; Primiget al., 2000; Deutschbaueret al., 2002). Interestingly, these studies show the number of regulatory genes that negatively affect sporulation is far lower than those that are activators of sporulation and moreover their mechanism of action is poorly studied. S. cerevisiae.SUB1 is one among negative regulators of sporulation(Deutschbaueret al., 2002). Global transcriptome of diploid yeast cells undergoing sporulation showed SUB1 transcripts are greatly reduced with time progression (Chu et al., 1998). To understand the role of SUB1 in sporulation, we generated deletion of both SUB1 alleles in the diploid S288c strain background and compared the kinetics of asci formation in this strain with that of the wild-type. We observed that cells lacking SUB1 exhibit ~5-fold increase in tetrad asci. Based on Eosin Y and Calcoflour White staining assays, we find no change in spore morphology in the mutant. Thus the increase in sporulation efficiency in sub1/sub1diploids is not accompanied by formation of defective spores. We validated the reduction in SUB1 transcript levels during sporulation in wild-type SK1 strain background. We also examined the Sub1 protein levels by epitope-tagging of the chromosomal SUB1 open reading frame and determining protein levels in this strain. We find that consistent with the data on transcript levels, Sub1-TAP tagged protein levels too decreased gradually on shift to sporulation medium. We created sub1alleles in diploids in the SK1 strain background and using this strain background we investigated Sub1 target genes and chose IME2 (early), SMK1, SPS2 (middle), DIT1, DIT2 (mid-late) and SPS100 (late) genes as representative sporulation genes. We observed that sub1∆/sub1∆cells have a significantly elevated expression of middle genes (SPS2 and SMK1) that followed normal induction kinetics i.e., 5 hours post transfer to sporulation medium. However, the expression levels or timing for other class of sporulation genes did not change in sub1∆strain as compared with the wild-type. In order to confirm these observations, we also studied the effects of over-expression of SUB1 from the GAL1 promoter by transforming the high copy plasmid. This was done in wild-type SK1 cells and the expression of sporulation genes were analyzed. We observed that expression of SMK1 and SPS2middle sporulation genes was reduced on over-expression of SUB1.We used the Sub1-TAP protein to assess if Sub1 directly regulates these genes by Chromatin immunoprecipitation assays. For these studies, we examined the recruitment of Sub1 to these loci through the time course of sporulation. In wild-type SK1 cells, Sub1 was to bound to middle sporulation genes and this was striking in cells at 5th hour post-induction of sporulation. These data establish that Sub1 directly associates with chromatin at these loci co-incident with the time points where expression levels of these changes is altered in cells lacking Sub1. Furthermore, to assess the role of Sub1 in other stress responses, such as pseudohyphae formation in response to nitrogen starvation, pheromone-induced agar invasion and secretory stress, we employed a genetic approach. Genetic interaction studies of SUB1 with RPB4, a subunit of RNA polymerase with functions in stress response and HOS2, a subunit of Set3 complex and a close homolog of mammalian HDAC3, reported to be involved in sporulation and secretory stress, were performed. Based on sporulation frequency and pseudohyphal formation in the double mutants we conclude that SUB1 is downstream of both these genes. Moreover, our results from assays of schmoo formation and pheromone-induced agar invasion suggest that SUB1 functionally interacts with HOS2. Study of domain architecture of Sub1 and homology to human PC4 Comparison of the S. cerevisiae Sub1 protein with its higher eukaryotic homologs showed that the N-terminal region of yeast Sub1 (32-105 aa) is highly conserved (Knauset al., 1996; Henry et al., 1996) with the 106-292 C -terminal amino acids being yeast-specific. We employed deletion analysis to generate partial Sub1 proteins and used them to understand the roles played by these domains in different phenotypes associated with Sub1. Our analysis of the localization of various Sub1-GFP fusion proteins shows that 146-172 aa in the C-terminal domain of Sub1 confers nuclear localization. Sporulation frequency analysis of the different domains of Sub1 suggests that both the N and C terminal domains are necessary for sporulation function of Sub1. The N terminal domain of yeast Sub1 shares homology with human PC4 and not surprisingly possesses ssDNA binding ability first attributed to human PC4 (Kaiser et al., 1995). In order to investigate whether the effects of SUB1 on kinetics of sporulation require its ssDNA binding function, we generated the sub1(Y66A) ssDNA binding mutant (Sikorskiet al., 2011) and over-expressed it in the S288c genetic background. We assessed sporulation efficiency of sub1∆/sub1∆cells over-expressing sub1(Y66A) mutant allele as compared to cells over-expressing wild-type SUB1. Interestingly, cells with over-expression of sub1(Y66A) have reduced sporulation efficiency that is equivalent to the levels achieved on over-expression of wild type SUB1. This data suggests that the ssDNA-binding ability of Sub1 is not important for its role in sporulation. Furthermore, we examined the ability of human PC4 to contribute to yeast sporulation process by complementation analysis. We observed that over-expression of PC4 complemented the phenotypes of sub1∆strain, suggesting that the function of Sub1/PC4 family is evolutionarily conserved. Studies on biochemical interactions of Sub1 with histone proteins Human PC4 is a chromatin-associated protein, present on metaphase chromosomes (Das et al., 2006). The short C-terminal domain of PC4(62-87 aa) interacts with core histones H3 and H2B in vitro and in vivo and this interaction mediates chromatin condensation. The homology between S. cerevisiaeSub1 (32-105 aa) and human PC4 (62-127 aa)is in the domain required for their DNA binding properties and coactivator functions, suggesting possible conservation in their interactions. We tested the interactions of yeast Sub1 with histone proteins by adopting both in vitro and in vivo interaction assays. We find recombinant Sub1 had strong interactions with rat and yeast histone H3in vitro. Moreover,Sub1 was found to interact with histone H2B, but not with H2A, in vivo, a binding specificity also shown by human PC4.Thus, we demonstrate conservation in the interaction of Sub1 with histone proteins.

Call number: LD2668 .T4 1979 F47 / Master of Science

Call number: LD2668 .T4 1955 B68 / Master of Science

Human papilloma virus (HPV) is a ubiquitous virus implicated in a growing list of cancers, particularly cervical cancer‐ the second most common cancer among women worldwide. Although persistent infection with high‐risk oncogenic HPVs such as types ‐16 or ‐18 is necessary, additional factors like co‐infection with other viruses can play a role in cancer progression. Protein‐protein interactions play a central role in the infection, survival and proliferation of the virus in the host. Although some interactions of HPV proteins are well characterised, it is essential to discover other key viral interactions to further improve our understanding of the virus and to use this knowledge for the development of newer biomarkers and therapeutics. The aim of this study was to systematically analyse the interactions of HPV‐16 proteins using yeast two‐hybrid (Y2H). To achieve this, a clone collection of the viral proteome was generated by recombinatorial cloning and three independent Y2H screens were performed: (i) Intra‐viral screen to identify interactions among the HPV‐16 proteins; (ii) Inter‐viral screen to identify interactions with proteins of Herpes Simplex Virus (HSV) which is suggested to be a co‐factor; and (iii) Virus‐host screen to identify novel cellular binding partners. The intra‐viral Y2H screen confirmed some of the previously known interactions and also identified binding of the E1 and E7 proteins. Deletion mutagenesis was performed to map the interaction domains to the amino‐terminal 92 amino acids of E1 and carboxy‐terminal CxxC domain of E7. Replication assays suggest a possible repression of E1‐mediated episomal replication by direct binding of E7. The inter‐viral Y2H screen identified interactions of HPV proteins with seventeen HSV‐1 proteins including transcriptional regulator ICP4 and neurovirulance factor ICP34.5. The biological relevance of these interactions in the context of co‐infection is discussed. The virus‐host screen performed against a human cDNA library identified 54 interactions, a subset of which was validated by biochemical pull‐down assays. The functional relevance of an interaction between E7 and a proto‐oncogene spermatogenic leucine zipper protein (SPZ1) was further investigated suggesting a role of SPZ1 in E7‐mediated cell proliferation. The work presented in this thesis identifies several novel interactions of HPV proteins. Future work will involve the in‐depth elucidation of biological relevance of these interactions. In particular, the interactions of E7 with E1 and SPZ1 are of great interest to improve our understanding of the life cycle and pathogenesis of the virus which can be applied for improved strategies of prevention and treatment of malignancies caused by HPV.

Thesis (MSc)--Stellenbosch University, 1990. / ENGLISH ABSTRACT: To remain competitive in the market place, the South African wine industry will have to direct well-planned yeast strain-development programmes. However, the winemaker can only benefit from the extensive biochemical and molecular information of the yeast cell and the impressive arsenal of genetic techniques available, if the wine industry defines its requirements in genetic terms. The successful application of these genetic and recombinant deoxyribonucleic acid (DNA) techniques in breeding programmes depends on the availability of rapid and reliable techniques to differentiate between parental and hybrid strains. Ten strains of Saccharomyces cerevisiae used for commercial production of wine in South Africa, were characterised by electrophoretic banding patterns of total soluble cell proteins, DNA restriction fragments and chromosomal DNA. Variations in the protein and DNA profiles of strains N6, N21, N66, N76, N95 and N97 were apparent in the number, position and intensity of the bands. Strains N93 and N181 originated from the same culture and, as expected, displayed the same characteristic protein, DNA restriction fragment and chromosomal banding patterns. Similar protein and DNA profiles were also obtained for killer strain N96 and strain N91. Strain N91 is a derivative of strain N96, cured of the K2 killer character. Results obtained by electrophoretic fingerprinting and karyotyping corresponded well, indicating that these techniques are valuable in the identification and quality control of industrial wine yeasts. The value of electrophoretic fingerprinting and karyotyping was also demonstrated in a breeding programme. The aim of this breeding programme was to obtain hybrids that combine the desired oenological characteristics of strains N76 and N96, and of strains N96 and N181. The protein banding patterns of hybrids USM21, USM22 and USM23 were identical and contained a combination of prominent unique bands present in the profiles of parental strains, N76 and N96H (N96H is a haploid derived from N96). The DNA restriction fragment profiles of hybrids USM21, USM22 and USM23 contained slight variations, whereas their profiles were quite different from those of their parental strains, N76 and N96H. The contour clamped homogeneous electric field (CHEF) karyotypes of hybrids USM21, USM22 and USM23 were identical but differed from those of their parental strains, N76 and N96H. The protein profiles of hybrid USM30 and its parental strains, N96H and N181, were similar, whereas their DNA restriction fragment banding patterns and CHEF karyotypes showed discrete differences. In conclusion, protein and DNA fingerprinting techniques were found to be valuable in selecting four hybrid killer strains after mass spore-cell mating. These four killer hybrids contain desirable oenological properties long sought after by the South African wine industry. Fermentation trials and evaluation of these hybrids were conducted independently by the Deparment of Oenology, University of Stellenbosch and by Stellenbosch Farmers' Winery and they have now been released for commercial wine production. / AFRIKAANSE OPSOMMING: Om mededingend in die handel te bly, sal die Suid-Afrikaanse wynbedryf weloorwoe gisras-ontwikkelingsprogramme moet loads. Die wynmaker sal egter slegs voordeel kan trek uit die omvattende biochemiese en molekul...Lre inligting oor die gissel en die indrukwekkende arsenaal van genetiese tegnieke wat beskikbaar is, indien die wynbedryf sy vereistes in genetiese terme definieer. Die suksesvolle toepassing van hierdie genetiese en rekombinante deoksiribonuklei"ensuur (DNA) tegnieke in telingsprogramme sal afhang van die beskikbaarheid van vinnige en betroubare tegnieke om tussen ouerlike en hibried-rasse te onderskei. Tien rasse van Saccharomyces cerevisiae wat vir kommersiele wynproduksie in Suid-Afrika gebruik word, is met behulp van elektroforetiese bandpatrone van totale oplosbare selprotei"ene, DNA-restriksiefragmente en chromosomale DNA gekarakteriseer. Variasies in die protei"en- en DNA-profiele van rasse N6, N21, N66, N76, N95 en N97 het geblyk uit die aantal, posisie en intensiteit van die bande. Rasse N93 en N181 het uit dieselfde kultuur ontstaan en het, soos verwag, dieselfde karakteristieke protei"en-, DNA-restriksiefragmenten chromosomale bandpatrone getoon. Soortgelyke protei"en en DNA profiele is ook vir killerras N96 en ras N91 verkry. Ras N91 is 'n variant van ras N96 wat die K2 killerkenmerk verloor het. Resultate wat met behulp van elektroforetiese vingermerking en kariotipering verkry is, het goed ooreengestem en dui daarop dat hierdie tegnieke waardevol is vir die identifisering en beheer van industriele giste. Die waarde van elektroforetiese vingermerking en kariotipering in telingsprogramme is ook gedemonstreer. Die doel van hierdie telingsprogram was om hibriede te kry waarin die gewenste kenmerke van rasse N76 en N96, en van rasse N96 en N181, gekombineer is. Die protei"en-bandpatrone van hibriede USM21, USM22 en USM23 was identies en het 'n kombinasie van prominente unieke bande, teenwoordig in die profiele van hul ourlike rasse, N76 en N96H (N96H is 'n haploi"de afstammeling van N96), bevat. Die DNArestriksiefragment- profiele van hibriede USM21, USM22 en USM23 toon geringe onderlinge verskille, maar hul profiele het wesenlik van die van hul ouerlike rasse, N76 en N96H, verskil. Die kontoergeklampde-homogene-elektriese-veld (CHEF) elektroforetiese kariotipes van hibriede USM21, USM22 en USM23 was identies, maar het verskil van die van hul ouerlike rasse, N76 en N96H. Die protei"enprofiele van hibried USM30 en sy ouerlike rasse, N96H en N181, was soortgelyk, terwyl hul DNA-restriksiefragment-bandpatrone en CHEF-kariotipes diskrete verskille getoon het. Ten slotte is gevind dat prote'ien- en DNAvingermerkingstegnieke waardevol was in die seleksie van vier hibried-killerrasse na massa spoor-sel paring. Hierdie vier killerhibriede beskik oor gewenste wynkundige eienskappe waarna die Suid-Afrikaanse wynbedryf reeds lank soek. Fermentasie-proewe en evaluering is onafhanklik deur die Departement Wynkunde, Universitiet van Stellenbosch en deur Stellenbosch-Boerewynmakery gedoen en hulle is nou vir kommersiele wynproduksie vrygestel.

Thesis(DScAgric.)--Stellenbosch University, 1932. / No Abstract Available

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: • The UPLC-MS/MS analyses and quantification of novel 5α-reduced steroids using response factors. • The kinetic characterisation of human steroid 5α-reductase type 1 (SRD5A1), expressed in HEK-293 cells, towards 11OHA4 and 11OHT and their keto derivatives by progress curve analysis. • The subcloning, transformation and functional expression of SRD5A1 in the yeast expression system, P. pastoris. • The conversion of 11OHA4 and 11OHT and their keto derivatives by SRD5A1 expressed in P. pastoris. • The endogenous enzymatic activity in P. pastoris towards the 5α-reduced metabolites in the 11OHA4- and alternate 5α-dione pathways. • The potential application of P. pastoris as a biocatalyst in the production of 5α- reduced C19 steroids. / AFRIKAANSE OPSOMMING: Hierdie ondersoek beskryf: • Die UPLC-MS/MS analise en kwantifisering van nuut-ondekte 5α-gereduseerde steroïede met behulp van responsfaktore. • Die kinetiese karakterisering van menslike steroïed 5α-reduktase tipe 1 (SRD5A1), uitgedruk in HEK-293 selle, vir 11OHA4 en 11OHT en hul ketoderivate deur middel van progressiekurwe-analise. • Die subklonering, transformasie en funksionele uitdrukking van SRD5A1 in die gis P. pastoris. • Die omsetting van 11OHA4 en 11OHT en hul ketoderivate deur SRD5A1 uitgedruk in P. pastoris. • Die omsetting van 5α-gereduseerde steroïede in die 11OHA4 en alternatiewe 5α-dioon paaie deur endogene ensieme in P. pastoris • ‘n Ondersoek na die toepassing van die gisuitdrukkingstelsel as ‘n moontlike OR potensiële biokatalis vir die produksie van 5α-gereduseerde C19 steroïede.

Thesis (MSc)--Stellenbosch University, 2003. / 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. / 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.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The grape and wine ecosystem contains fungi, bacteria and yeasts whose interactions contribute to the final wine product. While the non-Saccharomyces yeasts are dominant in the early stage of alcoholic fermentation, the later stage is always dominated by Saccharomyces cerevisiae. Although their presence in wine fermentation is often short-lived, the non-Saccharomyces yeasts are known to produce an array of extracellular hydrolytic enzymes which facilitate the extraction and release of aroma compounds, but might also play a role in microbial interactions. The present study aimed to investigate the microbial diversity of grape juice and to evaluate the potential of non-Saccharomyces yeasts to produce hydrolytic enzymes and display anti-fungal properties. To capture the microbial diversity, culture-dependent (plating) and –independent (Automated Ribosomal Intergenic Spacer Analysis (ARISA)) techniques were used in parallel. The fungal and bacterial ARISA displayed a wider range of operational taxonomic units (OTUs) in comparison to cultivation-based technique, demonstrating that ARISA is a powerful culture-independent technique applicable to ecological studies in wine. Some of the uncommon yeast isolates derived from our cultivation-based study were subjected to an enzymatic screening process. Hydrolases, such as chitinases, β-1,4-cellulases, β-1,3-1,6-glucanases, β-glucosidases, pectinases and acid proteases were specifically sought. Most of the yeast isolates exhibited chitinase, β-1,4-cellulase as well as β-1,3-1,6-glucanase activities. Only Metschnikowia chrysoperlae exhibited β-glucosidase activity. We also retrieved the partial chitinase gene sequences from M. chrysoperlae, Pichia burtonii, Hyphopichia pseudoburtonii that exhibited chitinase activity. Among the isolates, Pseudozyma fusiformata exhibited a strong antagonistic activity against the wine spoilage yeasts B. bruxellensis AWRI 1499 and B. anomalus IWBT Y105. Furthermore, we showed that the killer phenotype of P. fusiformata cannot be attributed to a viral encoded dsRNA. Finally, two metagenomic approaches were employed in an attempt to explore the indigenous microbiome in a more holistic manner, where we adopted whole metagenome Roche GS-FLX 454-pyrosequencing and construction of a fosmid library. The whole metagenome sequencing revealed a wide range of hydrolytic enzymes that showed homology to enzymes from different fungal and non-Saccharomyces yeast species. Moreover, the metagenomic library screening resulted in the retrieval of 22 chitinase and 11 β-glucosidase positive fosmid clones originating from yeasts. Two clones of interest, BgluFos-G10 and ChiFos-C21, were subjected to next generation sequencing. BgluFos-G10 revealed 2 ORFs exhibiting homology to glycosyl hydrolase family 16 proteins whereas no ORFs encoding chitinase enzymes could be identified in the ChiFos-C21 clone. However, all the potential ORFs identified exhibited homology to a gene cluster from Clavispora lusitaniae ATCC 42720, suggesting that the cloned DNA fragments belonged to a yeast species closely related to C. lusitaniae or members of the family Metschnikowiaceae. Overall, our study identified a variety of novel hydrolytic enzymes. However, retrieving the full gene sequences of these identified enzymes would be the immediate follow-up of our study. Moreover, the hydrolytic and antifungal activities exhibited by the yeast isolate could be of major interest in evaluating their potential as biocontrol agents against grapevine fungal pathogens and subsequently the wine spoilage yeasts. It would be interesting to evaluate as well the potential impact of these enzymes under wine making condition and could be our next step of investigation. / AFRIKAANSE OPSOMMING: Die druif en wyn ekosisteme bevat swamme, bakterië en giste en die interaksies van hierdie organismes dra by tot die finale wyn produk. Die nie-Saccharomyces giste is dominant in die vroeë stadium van die alkoholiese fermentasie, maar die latere fase word altyd gedomineer deur Saccharomyces cerevisiae. Alhoewel hulle teenwoordigheid in wyngistings gewoonlik kortstondig is, is die nie-Saccharomyces giste bekend vir die produksie van ‘n verskeidenheid ekstrasellulêre hidrolitiese ensieme wat die ekstraksie en vrylating van aroma komponente fasiliteer, en ook moontlik ‘n rol kan speel in mikrobiese interaksie. Hierdie studie beoog om die mikrobiese diversiteit van druiwesap te bestudeer en die potensiaal van nie-Saccharomyces giste te evalueer ten opsigte van die produksie van hidrolitiese ensieme, asook die demonstrasie van anti-swam eienskappe. Kweking-afhanklike (uitplating), asook –onafhanklike (Automatiese Ribosomale Intergeniese Spasieerder Analise (ARISA)) tegnieke is in parallel gebruik om die mikrobiese diversiteit te bepaal. Die swam en bakteriële ARISA het ‘n groter verskeidenheid van operasionele taksinomiese eenhede (OTUe) vertoon in vergelyking met die kweking-gebasseerde tegniek en dit demonstreer dat ARISA ‘n kragtige kweking-onafhanklike tegniek is, wat toepasbaar is in ekologiese studies van wyn . Sommige van die skaarser gisisolate, uit ons kweking -gebasseerde studie was vir ensiemaktiwiteite geskandeer. Daar is spesifiek gesoek vir hidrolases soos chitinases,β-1,4-sellulases, β-1,3-1,6-glukunases, β-glukosidases, pektinases en suur proteases. Die meeste gisisolate het chitinase,β-1,4-sellulase asook β-1,3-1,6-glukunase aktiwiteit vertoon. Slegs Metschinikowia chrysoperlae het β-glukosidase aktiwiteit vertoon. Ons het verder die gedeeltelike chitinase geensekwensies van M. chrysoperlae, Pichia burtonii en Hyphopichia pseudoburtonii wat chitinase aktiwiteit vertoon het, bepaal. Een isolaat, Pseudozyma fusiformata, het ‘n sterk antagonistiese aktiwiteit teenoor die wyn bederfgiste, Bretanomyces bruxellensis AWRI 1499 en B. anomalus IWBT Y105 vertoon. Verder het ons gewys dat die killer fenotipe van P. fusiformata nie gekoppel kan word aan’n viraal gekodeerde dsRNA nie. Ten laaste is twee metagenomiese benaderings, naamlik die volledige metagenoom Roche GS-FLX 454-pirovolgordebepaling en konstruksie van ‘n fosmied biblioteek, gebruik om die inheemse mikrobioom op ‘n meer holistiese wyse te bestudeer. Die volgordebepaling van die volledige metagenoom het ‘n wye verskeidenheid hidrolitiese ensieme aan die lig gebring wat homologie met ensieme van verskillende swamme en nie-Saccharomyces gisspesies getoon het. Verder het die skandering van die metagenomiese biblioteek die isolasie van fosmiedklone van gisoorsprong wat positief is vir chitinase aktiwiteit (22 klone) en β-glukosidase aktiwiteit (11 klone) tot gevolg gehad. Twee van hierdie klone, BgluFos-G10 en ChiFos-C21, is met volgende generasie volgordebepaling ontleed. BgluFos-G10 het twee oopleesrame (OLRe) wat homologie met glikosiel hidrolase familie 16 proteïene het, vertoon maar geen OLRe wat chitinase ensieme enkodeer kon in die ChiFos-C21 kloon geïdentifiseer word nie. Al die potensiële OLRe wat geïdentifiseer is, het homologie aan ‘n genepoel van Clavispora lusitaniae ATCC 42720 vertoon, wat daarop dui dat die gekloneerde DNS fragmente aan ‘n gisspesie behoort wat naverwant aan C. lusitaniae of lede van die Metschinikowiaceae familie is. In geheel gesien het ons studie ‘n verskeidenheid van nuwe hidrolitiese ensieme geïdentifiseer. Die bepaling van die volledige geenvolgordes van hierdie geïdentifiseerde ensieme sal die onmiddelike opvolg aksie van hierdie studie wees. Verder is die hidrolitiese en anti-swam aktiwiteite wat deur die gisisolate gedemonstreer is, van hoof belang, asook die evaluering van hulle potensiaal as biokontrole agente teen wingerd swampatogene en wyn bederfgiste. Dit sal ook interessant wees om die potensiële impak van hierdie ensieme onder wynmaakkondisies te bepaal, en dit kan dus ons volgende ondersoek stap wees.