As a first step toward improving creeping bentgrass (Agrostis palustris Huds.) via genetic engineering, this study was conducted to develop genetic transformation systems in creeping bentgrass. Establishment of embryogenic cell cultures is a prerequisite for crop improvement via genetic engineering. A protocol for initiating and maintaining embryogenic callus and suspension cultures in creeping bentgrass was developed by substantially modifying and combining a few existing protocols. A high frequency of plant regeneration was obtained following this protocol. Several factors affecting electroporation efficiency were studied using transient expression assay of the reporter uuid gene encoding B-glucuronidase (GUS). Increases in plasmid DNA resulted in increases in GUS activity. Maximal GUS activity was observed at field strength of 950 V/cm, protoplast density of 2 x 10⁶/ml, and KCl concentration of 125 mM in the electroporation buffer. Information obtained from this study facilitated optimization of electroporation conditions. To identify a 5’ regulatory sequence conferring a high level of transgene expression in creeping bentgrass, the effect of six different 5’ regulatory sequences on transient gene expression was studied in electroporated creeping bentgrass protoplasts. The cauliflower mosaic virus (CaMV) 35S promoter was least active; whereas the rice actin 1 gene 5’ sequence was most active among the six sequences tested. Ranked in order of activity (high to low), the other four 5’ sequences were: 1) the CaMV 35S promoter plus the maize alcohol dehydrogenase 1 gene (Adh1) intron 6; 2) the 5’ sequence of the maize ubiquitin gene (Ubi-1), 3) the maize Adh1 promoter and its intron 1, and 4) the 35S promoter plus the Adh1 intron 1. Stable transformation of creeping bentgrass was conducted via particle bombardment and electroporation using a plasmid, pZO1052, containing the reporter B-glucuronidase (uidA) gene and the selectable marker hygromycin phosphotransferase (hph) gene under the control of CaMV 35S promoter plus the maize Adh1 intron 6. Putative transformants were selected by culturing cells on medium containing hygromycin. Transgenic plants and calli were obtained following particle bombardment. The frequency of putative transformants was 4.6 hygromycin-resistant colonies per bombardment. Integration of the transgenes was confirmed by Southern blot hybridization. A high frequency of escapes, however, occurred in the transformant selection following electroporation, which resulted in inefficient transformant recovery. In this study, efficient genetic transformation systems using particle bombardment were established. Use of these systems will facilitate the improvement of creeping bentgrass. / Ph. D.

No description available.

Thesis (MSc)--Stellenbosch University, 2002. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: The winemaking process constitutes a unique ecological niche that involves the interaction of yeasts, lactic acid bacteria and acetic acid bacteria. Saccharomyces cerevisiae has established its importance as a wine yeast and also proven itself as a reliable starter culture organism. Its primary role is to convert the grape sugar into alcohol and, secondly, its metabolic activities result in the production of higher alcohols, fatty acids and esters, which are important flavour and aroma compounds that are essential for consistent and predictable wine quality. There is a growing consumer demand for wine containing lower levels of alcohol and chemical preservatives. Glucose oxidase (GOX) has received considerable research interest regarding its potential application in the wine industry to reduce alcohol levels and as a biocontrol agent. Several physical processes are used for the removal or reduction of alcohol in wine and some of them are sometimes used in combination. These processes tend to involve expensive equipment and can be intensive from a processing point of view. An alternative approach was introduced with the concept of treating grape must with GOX to reduce the glucose content of the must, and therefore produce a wine with a reduced alcohol content after fermentation. Due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialised wine yeast strains possessing a wide range of optimised, improved or novel oenological properties. The first and main objective of this study was to genetically engineer wine yeasts to produce wine with a reduced alcohol content. In order to do this, the structural glucose oxidase (gox) gene of Aspergillus niger was cloned into an integration vector (Ylp5) containing the yeast mating pheromone a-factor secretion signal (MFa1 s) and the phosphoglycerate kinase 1 gene promoter and terminator (PGK1PT). This PGK1p-MFa1sgox- PGKh gene cassette (designated GOX1) was introduced into a laboratory strain of S. cerevisiae (~1278). Results obtained indicated the production of biologically active glucose oxidase and showed that it is secreted into the culture medium. This would mean that the enzyme will convert the glucose to gluconic acid in the medium before the yeast cells are able to metabolise the glucose to ethanol. Microvinifications performed with Chardonnay grapes showed that the laboratory yeast starter cultures transformed with GOX1 were indeed able to reduce the total amount of alcohol in the finished product. The second objective of this study involved the potential application of GOX as a biocontrol agent. Screening was performed for wine spoilage microorganisms, such as acetic acid bacteria and lactic acid bacteria, using plate assays. The wine spoilage microorganisms tested formed different sized inhibition zones, indicating varying degrees of inhibition. The inhibition of some of the wine spoilage microorganisms was confirmed under a scanning electron microscope. The total collapse of the bacterial cell wáll could be seen and might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide (H202). The produced H202 leads to hyperbaric oxygen toxicity, a result of the peroxidation of the membrane lipid, and a strong oxidising effect on the bacterial cell, which is the cause of the destruction of basic molecular structures, such as nucleic acids and cell proteins. In this exciting age of molecular yeast genetics and modern biotechnology, this study could pave the way for the development of wine yeast starter culture strains for the production of wine with a lower alcohol content and reduced levels of chemical preservatives, such as sulphur dioxide. The use of genetically modified organisms (GMOs) within the wine industry is a limiting factor at present and credible means must be found to effectively address the concerns of traditionalists within the wine industry and the negative overreaction by some consumer groups. There is a vast potential benefit to the wine consumer and industry alike and the first recombinant wine products therefore should unmistakably demonstrate safe products free of potentially harmful compounds, and have organoleptic, hygienic and economic advantages for both the wine producer and consumer. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n ekologiese interaksie tussen gis, asynsuurbakterieë en melksuurbakterieë. Saccharomyces cerevisiae het homself alreeds bewys as 'n belangrike en betroubare inisiëringsgis in wyn. Die hoofdoel van die gis is om druifsuikers na etanol om te skakel. Tweedens lei die gis se metaboliese aktiwiteite tot die produksie van hoër alkohole, vetsure en esters, wat tot die konsekwente voorspelbare smaak en aromaverbindings in herhaalbare kwaliteit wyn bydra. Daar is 'n toenemende aanvraag na wyne met 'n laer alkoholinhoud en minder preserveermiddels. Glukoseoksidase (GOX) het heelwat navorsing in die wynindustrie uitgelok omdat dit gebruik kan word om die alkoholinhoud in wyn te verlaag, asook as 'n biologiese beheermiddel kan funksioneer. Daar is reeds sekere fisiese prosesse wat gebruik kan word om die alkohol in wyn te verwyder of te verminder. Sommige van hierdie prosesse word soms in kombinasie gebruik. Die nadeel is egter dat hierdie prosesse baie duur en intensief is, veral ten opsigte van prosessering. 'n Alternatief om die alkoholinhoud van wyn te verlaag, het egter na vore gekom toe daar voorgestel is om die mos met GOX te behandel. As gevolg van die veeleisende aard van moderne wynmaakpraktyke en gesofistikeerde wynmarkte, is daar 'n nimmereindigende soektog na meer gespesialiseerde wyngisrasse wat 'n wye reeks van geoptimiseerde en verbeterde, en selfs unieke, wynkundige einskappe bevat. Die hoofdoelwit van hierdie navorsingsprojek behels die genetiese manipulasie van 'n gisras sodat dit in staat is om wyn met 'n laer alkoholinhoud te produseer. Om hierdie doel te verwesentlik, is die strukturele glukoseoksidasegeen (gox) van Aspergillus niger in 'n integreringsvektor gekloneer. Transkripsie-inisiëring en -terminering is deur fosfogliseraatkinase-1-promotor en -termineerder (PGK1PT) bewerkstellig. Die a-spesifieke gisferomoon-a-faktor (MFa1 s) is gebruik om die uitskeiding van GOX uit die gis te bewerkstellig. Saam vorm bogenoemde die PGK1p-MFals-gox-PGKh-geenkasset, wat as GOX1 bekend is. GOX1 is na 'n labaratoriumras van S. cerevisiae (:E1278) getransformeer. Resultate dui aan dat biologies aktiewe GOX geproduseer en uitgeskei word. Dit beteken dat van die glukose in die medium reeds na glukoonsuur omgesit sal word voordat die gis dit kan begin benut en alkohol produseer. Kleinskaalse wynmaakprosesse wat met Chardonnay-druiwe en GOX-produserende labaratoriumgis uitgevoer is, het inderdaad tot laer alkoholpersentasies gelei. Die tweede doelwit van die navorsingsprojek was om te bepaal of GOX die potensiaal as biologiese beheermiddel het. Daar is ondersoek ingestel na sekere wynbederfsorganismes soos asynsuur- en melksuurbakterieë en die inhibisie van die organismes is op agarplate gemonitor. Verskillende grade van inhibisie, soos die grootte van die inhibisiesone, was sigbaar vir die verskillende wynbederfsorganismes wat getoets is. Die inhibiese van sekere wynbederfsorganismes is ook met behulp van 'n skandeerelektronmikroskoop bevestig. Die totale ineenstorting van die bakteriële selwand was sigbaar en kan verklaar word deur die teenwoordigheid van waterstofperoksied (H202). Laasgenoemde is 'n byproduk van die laaste metaboliese reaksie en staan as 'n antimikrobiese middel bekend. Die byproduk (H202) gee aanleiding tot hiperbariese suurstoftoksisiteit, 'n gevolg van die peroksidasie van membraanlipiede en 'n sterk oksiderende effek t.o.v. die bakteriële selwand. Dit lei tot die vernietiging van die basiese molekulêre strukture, soos die nukleïensure en selproteïene. Tydens hierdie opwindende era van molekulêre gisgenetika en biotegnologie kan hierdie navorsing die fondament lê vir die ontwikkeling van 'n wyngiskultuur wat in staat is om wyn met 'n laer alkoholinhoud te produseer. Die gebruik van geneties gemanupileerde organismes (GMO's) in die wynbedryf is egter nog 'n beperkende faktor. 'n Geloofwaardige manier moet dus gevind word om die bekommernisse van tradisionaliste, asook die negatiewe oorreaksies van sommige verbruikers, aan te spreek en hok te slaan. Daar is groot potensiaal en voordele vir beide die verbruiker en industrie. Dit is dus belangrik dat die eerste rekombinante wynprodukte wat die mark betree, veilig en vry van potensieel skadelike verbindings is, asook organoleptiese, higiëniese en ekonomiese voordele toon te opsigte van beide die wynprodusent en gebruiker.

Ho, Wing Ho. / On t.p. "beta" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 157-183). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.vi / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xv / List of Figures --- p.xvii / List of Abbreviations --- p.xxiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.4 / Chapter 2.1 --- Vitamin A --- p.4 / Chapter 2.1.1 --- Genral and properties --- p.4 / Chapter 2.1.2 --- Biological importance of vitamin A --- p.6 / Chapter 2.1.3 --- Dietary source of vitamin A --- p.12 / Chapter 2.1.3.1 --- Plant-derived provitamin A and animal-derived vitamin A --- p.12 / Chapter 2.1.3.2 --- Dependence on the plant-derived provitamin A by the poor --- p.14 / Chapter 2.1.3.2.1 --- Plant-derived provitamin A --- p.14 / Chapter 2.1.3.2.1.1 --- General and properties --- p.14 / Chapter 2.1.3.2.1.2 --- Biosynthesis of provitamin A in plants --- p.17 / Chapter 2.1.3.2.1.2.1 --- Assembly of C40 backbone … --- p.17 / Chapter 2.1.3.2.1.2.2 --- Desaturation and cyclization --- p.26 / Chapter 2.1.3.2.1.2.3 --- Oxygenation --- p.29 / Chapter 2.1.3.2.1.2.4 --- Carotenogenic enzymes --- p.31 / Chapter 2.1.4 --- Metabolism of dietary vitamin A and provitamin A in human system --- p.35 / Chapter 2.1.4.1 --- Digestion and absorption --- p.35 / Chapter 2.1.4.2 --- Biocon version --- p.37 / Chapter 2.1.4.2.1 --- "Beta, beta '-carotene 15, 15'-monooxygenase (BCMO)" --- p.40 / Chapter 2.1.4.3 --- "Transport, uptake and storage" --- p.43 / Chapter 2.1.4.4 --- Provision or excretion --- p.46 / Chapter 2.2 --- Vitamin A deficiency (VAD) --- p.48 / Chapter 2.2.1 --- Green revolution --- p.48 / Chapter 2.2.2 --- Rice as the major staple food for feeding the poor --- p.49 / Chapter 2.2.3 --- Provitamin A content in processed rice seeds --- p.49 / Chapter 2.2.4 --- Symptoms of VAD --- p.51 / Chapter 2.2.5 --- Global prevalence of VAD --- p.53 / Chapter 2.3 --- Previous efforts for dealing with the deficiency --- p.55 / Chapter 2.3.1 --- The key for dealing with the deficiency --- p.55 / Chapter 2.3.2 --- Selective plant breeding --- p.55 / Chapter 2.3.3 --- Supplementation and post-harvesting fortification --- p.56 / Chapter 2.3.4 --- Bio-fortification by genetic engineering --- p.57 / Chapter 2.3.4.1 --- Advantages of genetic engineering --- p.57 / Chapter 2.3.4.1.1 --- Genetic engineering of non-cereal crops --- p.58 / Chapter 2.3.4.1.2 --- Genetic engineering of cereal crops --- p.62 / Chapter 2.3.4.1.2.1 --- Golden Rice 1 --- p.62 / Chapter 2.3.4.1.2.2 --- Golden Rice 2 --- p.64 / Chapter 2.4 --- Motivation for striking forward --- p.67 / Chapter 2.4.1 --- Recommended Dietary Amount of vitamin A --- p.67 / Chapter 2.4.2 --- Factors affecting the bioefficacy of provitamin A in human body --- p.68 / Chapter 2.4.2.1 --- Bioavailability --- p.68 / Chapter 2.4.2.2 --- Bioconvertibility --- p.69 / Chapter 2.4.2.3 --- Health and nutritional status --- p.71 / Chapter 2.4.3 --- Further improvement for dealing with the deficiency --- p.73 / Chapter 2.5 --- Hypothesis --- p.75 / Chapter Chapter 3 --- Materials and Methods --- p.78 / Chapter 3.1 --- Chemicals --- p.78 / Chapter 3.2 --- Bacterial strains --- p.78 / Chapter 3.3 --- Transient expression of BCMOs in plant system --- p.79 / Chapter 3.3.1 --- Choice of BCMOs --- p.79 / Chapter 3.3.2 --- Plasmids and genetic material --- p.79 / Chapter 3.3.3 --- Construction of chimeric genes for transient expression --- p.82 / Chapter 3.3.4 --- Microprojectile bombardment and GUS assay --- p.83 / Chapter 3.4 --- Construction of chimeric genes for rice co-transformation --- p.84 / Chapter 3.4.1 --- Choice of carotenogenic genes --- p.84 / Chapter 3.4.2 --- Choice of promoters --- p.84 / Chapter 3.4.3 --- Necessities and choice of transit peptide (TP) --- p.85 / Chapter 3.4.4 --- Arrangement of chimeric gene-cassettes --- p.86 / Chapter 3.4.5 --- Plasmids and genetic materials --- p.87 / Chapter 3.4.6 --- Construction of chimeric gene expressing PSY and PDS coordinately --- p.87 / Chapter 3.4.7 --- "Construction of chimeric gene expressing PSY, PDS and TP equipped CHBCMO coordinately" --- p.92 / Chapter 3.4.8 --- "Construction of chimeric gene expressing PSY, PDS and TP equipped ZEBCMO coordinately" --- p.98 / Chapter 3.4.9 --- Construction of chimeric gene expressing ZDS and LYCB coordinately --- p.103 / Chapter 3.4.10 --- Confirmation of sequence fidelity --- p.108 / Chapter 3.5 --- Rice co-transformation --- p.109 / Chapter 3.5.1 --- Plant materials --- p.109 / Chapter 3.5.2 --- Preparation of Agrobacterium tumefaciens --- p.109 / Chapter 3.5.3 --- Agrobacterium mediated co-transformation --- p.110 / Chapter 3.5.3.1 --- Callus induction from mature rice seeds --- p.110 / Chapter 3.5.3.2 --- Callus induction from immature rice seeds --- p.110 / Chapter 3.5.3.3 --- "Co-cultivation, selection and regeneration" --- p.111 / Chapter 3.6 --- Detection of transgene expression --- p.112 / Chapter 3.6.1 --- Detection at DNA level --- p.112 / Chapter 3.6.1.1 --- Genomic DNA extraction --- p.112 / Chapter 3.6.1.2 --- PCR screening --- p.112 / Chapter 3.6.1.3 --- Synthesis of DIG-labeled DNA probes --- p.114 / Chapter 3.6.1.4 --- Southern blot analysis --- p.115 / Chapter 3.6.2 --- Detection at RNA level --- p.116 / Chapter 3.6.2.1 --- Total RNA extraction --- p.116 / Chapter 3.6.2.2 --- Northern blot analysis --- p.116 / Chapter 3.6.3 --- Detection at product level --- p.117 / Chapter 3.6.3.1 --- Phenotypic identification --- p.117 / Chapter 3.6.3.2 --- HPLC analysis --- p.117 / Chapter 3.6.3.2.1 --- Extraction of total carotenoids and retinoids --- p.117 / Chapter 3.6.3.2.2 --- HPLC identification --- p.118 / Chapter 3.6.3.2.3 --- HPLC quantification --- p.118 / Chapter Chapter 4 --- Results --- p.119 / Chapter 4.1 --- Transient expression of BCMOs in plant system --- p.119 / Chapter 4.1.1 --- Construction of chimeric genes for transient expression --- p.119 / Chapter 4.1.2 --- Microprojectile bombardment and GUS assay --- p.120 / Chapter 4.2 --- Construction of chimeric genes for rice co-transformation --- p.121 / Chapter 4.3 --- Rice co-transformation --- p.123 / Chapter 4.3.1 --- Callus induction from mature and immature rice seeds --- p.123 / Chapter 4.3.2 --- "Co-cultivation, selection and regeneration" --- p.124 / Chapter 4.4 --- Detection of transgene expression --- p.126 / Chapter 4.4.1 --- Detection at DNA level --- p.126 / Chapter 4.4.1.1 --- PCR screening --- p.126 / Chapter 4.4.1.2 --- Southern blot analysis --- p.129 / Chapter 4.4.2 --- Detection at RNA level --- p.133 / Chapter 4.4.2.1 --- Northern blot analysis --- p.133 / Chapter 4.4.3 --- Detection at product level --- p.135 / Chapter 4.4.3.1 --- Phenotypic identification --- p.135 / Chapter 4.4.3.2 --- HPLC identification --- p.137 / Chapter 4.4.3.3 --- HPLC quantification --- p.147 / Chapter Chapter 5 --- Discussion --- p.150 / Chapter Chapter 6 --- Conclusion --- p.156 / References --- p.157

A majority of methods for identifying sequences in the human genome involve target sequence amplification through PCR. This work presents novel methods for amplifying circularized DNA and presents solutions for some major limitations of PCR. We have developed a novel method to amplify circularized DNA molecules based on a serial rolling-circle replication reaction, called circle to circle amplification (C2CA). Amplified DNA circles can be detected in array-based analyses or in real-time using molecular beacons. The amplification mechanism allows higher precision in quantification than in exponential amplification methods like PCR, and more products can be generated than in PCR. A major limitation of PCR is that amplification artifacts arise when large numbers of specific primer pairs are simultaneously added to a reaction. We have developed a solution to this problem that enables multiplex PCR amplification of specific target sequences without producing amplification artifacts. The procedure is based on oligonucleotide constructs, called selectors. The selectors identify defined target nucleic acid sequences, and they act as ligation templates to direct circularization of these targets. The selectors contain a general primer-pair motif that allows the circularized targets to be amplified in multiplex using a universal PCR primer pair. We also developed a computer program, PieceMaker, that finds an optimal design of selector probes for a given selector application. We demonstrate the method by performing a 96-plex PCR of specific DNA sequences with high success-rate and reproducibility.

Coniothyrium zuluense is the causal agent of a serious Eucalyptus stem canker disease in South Africa (Wingfield et al., 1997). Eucalypts are the most important hardwood plantations in the world, and in South Africa these hardwoods occupy approximately 1.5 million hectares of plantation area, an area that is soon to be increased by an additional 600 000 hectares. As exotics, Eucalyptus plantations are constantly exposed to infection by fungal pathogens such as C. zuluense, which by secreting cell-¬wall degrading enzymes contribute to the degradation of plant cell walls and subsequent reduction and in the quality of timber produced. This ultimately affects the South African paper, pulp and timber industries. Selection of resistant clones through traditional breeding methods is the most common method currently employed in overcoming the problem of fungal infection. The genetic manipulation of Eucalyptus trees for enhanced resistance to fungal diseases is an alternative to the time-consuming and tedious approach of conventional breeding. The identification of several antifungal proteins, particularly polygalacturonase-inhibiting proteins (PGIPs) from various plant species including Eucalyptus, lead to the hypothesis that over-expression of these proteins could potentially reduce pathogen attack. However, prior to the expression of PGIPs in plants, isolation and molecular characterization of these genes are required. The aims of this study were therefore (l) to clone and characterize the complete Eucalyptus grandis pgip gene, (2) to transform Nicotiana tabacum (tobacco) plants with the E. grandis pgip gene and (3) to test for inhibition of C. zuluense PGs by PGIPs extracted from transgenic tobacco plants. This forms the first step towards the generation of E. grandis clones that are more disease tolerant. A review of the role of fungal endopolygalacturonases and polygalacturonase¬inhibitors in plant-pathogen interactions are presented in chapter I. Strategies employed to isolate and characterize pgip genes from a range of plant species are highlighted and the importance ofPGIPs in disease resistance is discussed. In chapter 2, the molecular cloning and characterization of the E. grandis pgip gene is discussed. The work presented in this chapter is a follow up on work previously conducted by Chimwamurombe (2001). Previously, a partial Eucalyptus pgip gene sequence was obtained with the use of degenerate oligonucleotide primers. In this study, the complete Eucalyptus pgip gene was obtained through the employment of genome walking strategies. Transformation of Nicotiana tabacum cv LA Burley plants with the Eucalyptus pgip gene and the molecular characterization of transgenic tobacco plants is discussed in chapter 3. The transformation and expression of foreign genes in tobacco plants is a well-established protocol, making tobacco the most appropriate candidate plant for assessing the functionality of the plant transformation construct. The production of endopolygalacturonases from virulent C. zuluense isolates and the subsequent PGIP assays conducted to determine levels of PG inhibition are included in this chapter. This thesis consists of three independent chapters representing studies on the molecular characterization of an E. grandis pgip gene and focusing on the potential for inhibition of PGs produced by C. zuluense by Eucalyptus PGIP extracted from transgenic tobacco plants. Repetition of certain aspects in the individual chapters has been unavoidable and the thesis is presented following a uniform style. / Dissertation (MSc)--University of Pretoria, 2003. / Genetics / Unrestricted

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The aim of this study was toe stablish a reliable protocol for the production 0 f transgenic pearl millet as this will open new avenues for augmenting the gene pool of this crop. This was achieved by identifying a highly regenerabie genotype and optimisation of a tissue culture system, and biolistic protocol f or stable integration of selected transgenes. Both a negative, herbicide resistance selectable marker gene, bar, and a positive selectable marker gene, manA, were individually introduced in order to identify and establish a reliable transformation protocol. The optimised transformation protocol was then used to introduce an antifungal gene in the genome of pearl millet to enhance resistance to the biotrophic fungus Sclerospora graminicola. S. graminicola, an obligate oomycetous fungal phytopathogen, is the causal agent of downy mildew in pearl millet plants and a major constraint in the production of pearl millet. A single component of antifungal resistance was introduced into the genome of pearl millet, as preliminary work towards determining its role in the total plant defence system. The approach chosen was to introduce a hydrolytic enzyme, 13-1,3- glucanase, from Trichoderma atroviride (formerly T. harzianum), a soil-borne filamentous fungus, capable of parasitizing several plant pathogenic fungi. It was anticipated that introducing this glucanase gene from T. atroviride which degrades glucan in the fungal cell walls, would significantly contribute to the improvement of resistance against downy mildew. Constructs were prepared containing the gene (gluc78) encoding a 78 kDa beta-1,3- glucanase. The constructs were prepared containing the gluc78 gene driven either by a strong constitutive promoter (ubiquitin promoter, exon and intron) or a wound inducible promoter, the potato proteinase inhibitor ilK gene promoter. The wound inducible promoter includes either an AMV leader' sequence or the rice Act1 intron to obtain higher expression levels in the monocotyledonous plant. The transformation efficiency using the particle inflow gun and the herbicide resistance gene, bar, was improved from 0.02% on a MS based medium, to 0.19 or 0.72% with manA as selectable marker gene on MS or L3 based medium, respectively. However, individual experiments, introducing manA as selectable marker gene, resulted in frequencies of 1.2 and 3%. This translated to one transformation event per plate, which contains on average 31-35 pre-cultured immature zygotic embryos. This is the first report of t he successful introduction and expression of a 13-1,3-glucanase encoding gene from a biocontrol fungus not only under constitutive expression but also under wound inducible expression in a plant. Optimisation of genetic engineering of pearl millet, a cereal crop recalcitrant to transformation, and the introduction of an antifungal transgene, was accomplished in this study. Initial results hint that expression of this transgene enhances resistance to S. graminicola. / AFRIKAANSE OPSOMMING: Die doel van die studie was om 'n betroubare genetiese transformeringsprotokol vir pêrel manna te ontwikkel. Hiervoor moes eerstens 'n regenereerbare genotipe geidentifiseer word. Twedens moes 'n betroubare weefselkultuur en biolistiese transformeringssisteem ontwikkel word. Beide die onkruiddoder bestandheidsgeen, bar, en 'n positiewe selektiewe geen, manA, is vir die doel van die projek onafhanklik in die genoom van pêrel manna in gekloneer. Die optimale sisteem is vervolgens aangewend om 'n geen wat potensieël verbeterde bestandheid teen die biotrofiese swam Sclerospora graminicola wat donsige meeldou by plante veroorsaak, in pêrel manna in te kloneer. 'n Enkele komponent van bestandheid is in die genetiese material van pêrel manna in gekloneer as inleidende werk om die rol van hierdie geen in die totale verdedigingsisteem te bepaal. Die benadering wat gekies was, behels die klonering van 'n hidrolitiese ensiem 13-1,3-glukanase, van Trichoderma atroviride (voorheen T. harzianum), 'n grondgedraagde swam, wat op 'n aantal ander plantpatogene fungus kan parasiteer. Die verwagting is dat klonering van hierdie 13- 1,3-glukanase geen van T. atroviride wat die glukaan verteer in die selwande van swamme, 'n groot verbetering tot die bestandheid teen donsige meeldou sal meebring. Konstrukte is voorberei wat die gluc78 geen bevat wat kodeer vir die 78 kDa beta-1,3-glukanase protein. Die konstrukte wat voorberei is bevat die gluc78 geen geinduseer deur of 'n sterk konstituwe promoter (ubiquitin promoter, exon en intron) of deur 'n wond geinduseerde promoter, die aartappel proteinase inhibeerder ilK geen promoter. Hierdie promoter word gevolg deur of 'n AMV leier volgorde of die rys Act1 intron om verhoogde uitdruk vlakke in monokotiele plante te verseker. As die partikel invloei geweer in kombinasie met die onkruiddoderbestandheidsgeen gebruik word, was die doeltreffendheid van transformasie 0.02% op 'n MS gebasseerde groeimedium. 'n Transformasie doeltreffendheid van onderskeidelik 0.19 en 0.72% is verkry wanneer die manA as selektiewe geen gebruik is op MS of L3 gebasseerde medium. Twee individual eksperimente, waar die manA geen as selektiewe geen gebruik is, het gelei tot 'n transformasie doeltreffendheid van 1.2 of 3%. Dit gee 'n gemiddelde van een transformasie per plaat wat 31 tot 35 voorafgekweekte onvolwasse embrios bevat. Hierdie is d ie eerste verslag van d ie suksesvolle klonering en uitdrukking van 'n 13-1,3-glukanasekoderende geen van 'n swam wat as 'n biologiese beheeragent gebruik word. Hierdie is nie alleenlik onder konstitutiewe uitdrukking nie, maar ook 0 nder wond g einduseerde u itdruk in' n p lant. In hierdie studie is die 0 ptimisering van genetiese verbetering van pêrel manna, 'n graan gewas wat gehard is teen transformasie, deur die klonering van 'n bestandheidsgeen in die genoom van hierdie gewas gedoen. Aanvanklike resultate dui daarop dat die uitdruk van hierdie geen lei tot verbeterde bestandheid teen S. graminicola.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2005. / Biomass is the sole foreseeable sustainable source of organic fuels, chemicals and materials. It is a rich and renewable energy source, which is abundant and readily available. Primary factors motivating the use of renewable enrgy sources include the growing concern over global climate change and the drastic depletion of non-renewable resources. Among various forms of biomass, cellulosic feedstocks have the greatest potential for energy production from. The biggest technological obstacle to large-scale utilisation of cellulosic feedstocks for the production of bioethanol as a cost-effective alternative to fossil fuels is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic biomass. A promising strategy to overcome this impediment involves the production of cellulolytic enzymes, hydrolysis of biomass and fermentation of resulting sugars to ethanol in a single process step via a single microorganism or consortium. Such “consolidated bioprocessing” (CBP) offers very large cost reductions if microorganisms, such as the yeast Saccharomyces cerevisiae, can be developed that possess the required combination of efficient cellulose utilisation and high ethanol yields. Cellulose degradation in nature occurs in concert with a large group of bacteria and fungi. Cellulolytic microorganisms produce a battery of enzyme systems called cellulases. Most cellulases have a conserved tripartite structure with a large catalytic core domain linked by an O-glycosylated peptide to a cellulose-binding domain (CBD) that is required for the interaction with crystalline cellulose. The CBD plays a fundamental role in cellulose hydrolysis by mediating the binding of the cellulases to the substrate. This reduces the dilution effect of the enzyme at the substrate surface, possibly by helping to loosen individual cellulose chains from the cellulose surface prior to hydrolysis. Most information on the role of CBDs has been obtained from their removal, domain exchange, site-directed mutagenesis or the artificial addition of the CBD. It thus seems that the CBDs are interchangeable to a certain degree, but much more data are needed on different catalytic domain-CBD combinations to elucidate the exact functional role of the CBDs. In addition, the shortening, lengthening or deletion of the linker region between the CBD and the catalytic domain also affects the enzymatic activity of different cellulases. Enzymes such as the S. cerevisiae exoglucanases, namely EXG1 and SSG1, and the Saccharomycopsis fibuligera β-glucosidase (BGL1) do not exhibit the same architectural domain organisation as shown by most of the other fungal or bacterial cellulases. EXG1 and SSG1 display β-1,3-exoglucanase activities as their major activity and exhibit a significant β- 1,4-exoglucanase side activity on disaccharide substrates such as cellobiose, releasing a free glucose moiety. The BGL1 enzyme, on the other hand, displays β-1,4-exoglucanase activity on disaccharides. In this study, the domain engineering of EXG1, SSG1 and BGL1 was performed to link these enzymes to the CBD2 domain of the Trichoderma reesei CBHII cellobiohydrolase to investigate whether the CBD would be able to modulate these non-cellulolytic domains to function in cellulose hydrolysis. The engineered enzymes were constructed to display different modular organisations with the CBD, either at the N terminus or the C terminus, in single or double copy, with or without the synthetic linker peptide, to mimic the multi-domain organisation displayed by cellulases from other microorganisms. The organisation of the CBD in these recombinant enzymes resulted in enhanced substrate affinity, molecular flexibility and synergistic activity thereby improving their ability to act and hydrolyse cellulosic substrates, as characterised by adsorption, kinetics, thermostability and scanning electron microscopic (SEM) analysis. The chimeric enzyme of CBD2-BGL1 was also used as a reporter system for the development and efficient screening of mutagenised S. cerevisiae strains that overexpress CBD-associated enzymes such as T. reesei cellobiohydrolase (CBH2). A mutant strain WM91 was isolated showing up to 3-fold more cellobiohydrolase activity than that of the parent strain. The increase in the enzyme activity in the mutant strain was found to be associated with the increase in the mRNA expression levels. The CBH2 enzyme purified from the mutant strain did not show a significant difference in its characteristic properties in comparison to that of the parent strain. In summary, this research has paved the way for the improvement of the efficiency of the endogenous glucanases of S. cerevisiae, and the expression of heterologous cellulases in a hypersecreting mutant of S. cerevisiae. However, this work does not claim to advance the field closer to the goal of one-step cellulose processing in the sense of technological enablement; rather, its significance hinges on the fact that this study has resulted in progress towards laying the foundation for the possible development of efficient cellulolytic S. cerevisiae strains that could eventually be optimised for the one-step bioconversion of cellulosic materials to bioethanol.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Cellulose and hemicellulose represents the two most abundant groups of renewable polysaccharides known to man. Apart from their presence in plant material, they also contribute to a significant portion of inexpensive readily available material, such as wastes and bypro ducts from forestry / agricultural origin. The chemical composition of plant material varies, but the biomass content consists of approximately 75% carbohydrate polymers (cellulose and hemicellulose) and 25% lignin. The enzymes required for the degradation of cellulose and hemicellulose are collectively called cellulases and hemicellulases. These enzymes have a broad spectrum of industrial applications including the production of fuel ethanol through fermentations, reducing the amount of chlorine required for bleaching in the pulp and paper industry, increasing dough volume in the baking industry, improving digestion and nutritional value of animal feed, increasing clarification and enhancing the filterability of wine, beer and fruit juice, etc. Therefore, a large potential market exists for cellulases and hemicellulases provided their production is economical and the product, authentic. Aspergilli occur in a wide variety of habitats including soil, stored food and feed products and decaying vegetation. The advantages for using A. niger as host for heterologous enzyme production include good protein secretion, industrial fermentation technology dating as far back as 1919, being a non-pathogenic fungus with GRAS status, no special substrate or cultivation requirements, FDA approval of numerous enzymes (homologous and heterologous) produced, etc. In this study an Aspergillus expression vector was constructed using the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdp) of A. niger and the glucoamylase terminator (glaAT) of Aspergillus awamori. The cDNA copies of the eg! and xyn2 genes of Trichoderma reesei, cbhl-4 of Phanerochaete chrysosporium, man! of Aspergillus aculeatus and xyn3 of Aspergillus kawachii were introduced into the expression vector, respectively. All the plasmids were co-transformed with plasmid p3SR2 to A. niger and transformants selected for stable plasmid integration into the genome of the host. The recombinant enzymes EgI, Xyn2, Cbhl-4, Man! and XynC were successfully expressed and secreted at activity levels of 2300, 8000, 500, 6000 and 900 nkatlml, respectively. The enzymes were produced as functional entities and were subsequently characterized. The EgI, Xyn2 and ManI were evaluated as feed additives for the possible use in the animal feed industry. Improved biomass gain was observed with in vivo studies on poultry. With the possible mass production of heterologous enzymes in mind, a simple medium had to be devised for their inexpensive production. Molasses medium (available from the South African sugar industry) was therefore evaluated and the cultivation conditions optimized for it's possible use as cultivation substrate for A. niger. The evaluation was done on the grounds of EgI and Xyn2 activity produced which was monitored over time. This study highlighted the possible use of A. niger for the heterologous production of enzymes, the use of industrial substrate for cultivation and paved the way for the high level expression of industrially important genes at low cost and a positive environmental impact. / AFRIKAANSE OPSOMMING: Sellulose en hemisellulose verteenwoordig die twee vollopste herwinbare polisakkariede bekend. Behalwe vir hul teenwoordigheid in plantmateriaal, dra hulle ook by tot 'n beduidende fraksie van goedkoop, maklik bekombare materiaal soos afval- en byprodukte van bosbou I landbou oorsprong. Soos te verwagte, varieër die chemiese samestelling van die plantmateriaal, maar die biomassa-inhoud bestaan uit naastenby 25% lignien en 75% koolhidraatpolimere (sellulose and hemicellulose). Die ensieme benodig vir die afbraak van sellulose en hemisellulose staan gesamentlik as sellulases en hemisellulases bekend. Hierdie ensieme het 'n breë spektrum van industriële toepassings insluitende die produksie van brandstofalkohol d.m.v. fermentasies, vermindering in die hoeveelheid chloor benodig vir die bleikproses in die pulp-en-papier industrie, toename in deegvolume in die bakkersindustrie, verbetering van verteerbaarheid en verhoging van voedingswaarde van dierevoer, toename in verheldering en verbeterde filtreerbaarheid van wyn, bier en vrugtesap, ens. Dus bestaan daar 'n groot potensiële mark vir sellulases en hemisellulases, mits hul produksie ekonomies en die produk outentiek is. Aspergilli kom in 'n wye verskeidenheid van omgewings voor, insluitende grond, gestoorde voedsel- en voerprodukte asook ontbindende plante materiaal. Die voordele vir die gebruik van A. niger as gasheer vir heteroloë ensiemproduksie sluit in 'n goeie proteïen produseerder, industriële fermentasietegnologie dateer sover terug as 1919, 'n nie-patogeniese fungus met GRAS-status, benodig geen spesiale substrate of kwekingskondisies nie, FDA goedkeuring vir 'n groot aantal ensieme (homoloog sowel as heteroloog) wat reeds geproduseer word, ens. In hierdie studie is 'n Aspergillus uitdrukkingsvektor gekonstrueer deur van die konstitutiewe gliseraldehied-3-fosfaat dehidrogenase promoter (gpdp) van A. niger en die glukoamilase termineerder (glaAT) van Aspergillus awamori gebruik te maak. Die cDNA kopiee van die die eg! en xyn2 van Trichoderma reesei, cbhl-4 van Phanerochaete chrysosporium, man! van Aspergillus aculeatus en die xynC van Aspergillus kawachii was onderskeidelik na die uitdrukkingsplasmied oorgedra. Alle plasmiede is gesamentlik met die p3 SR2 plasmied na A. niger getransformeer en vir stabiele integrasie in die gasheergenoom geselekteer. Die rekombinante ensieme Egl, Xyn2, Cbhl-4, Manl en Xyn3 is suksesvol uitgedruk en teen aktiviteitsvlakke van 2300, 8000, 500, 6000 en 900 nkat/ml, onderskeidelik uitgeskei. Die ensieme is as funksionele entiteite geproduseer en vervolgens gekaraktiriseer. Die Egl, Xyn2 en Manl is as voertoevoegings vir die moontlike gebruik in die dierevoerindustrie geëvalueer. Verbeterde biomassa toename is in die in vivo studie op pluimvee waargeneem. Met die moontlikheid van grootskaalse heteroloë ensiemproduksie in gedagte, moes 'n eenvoudige substraat vir hul goedkoop produksie gevind word. Molasse medium (verkrygbaar vanaf die Suid Afrikaanse suiker industrie) was derhalwe geëvalueer en die kwekingskondisies geoptimiseer vir die moontlike gebruik as kwekingssubstraat vir A. niger. Vir die evaluasie is die Egl en Xyn2 aktiwiteite onder verskillende toestande geproduseer en oor tyd gemonitor. Hierdie studie beklemtoon die moontlike gebruik van A. niger vir heteroloë produksie van ensieme, die gebruik van industriële substrate as kwekingsmedium en baan die weg vir ekonomiese, hoëvlakuitdrukking van industrieelbelangrike ensieme met 'n positiewe implikasie op die omgewing.

Thesis (PhD)--University of Stellenbosch, 1999. / ENGLISH ABSTRACT: Hemicellulose, consisting mainly of xylan, ranks after cellulose, as the most abundant group of renewable polysaccharides in agricultural biomass. Xylan is a complex polymer consisting of a β D 1,4 linked xylopyranoside backbone, which may contain substituents. Enzymatic hydrolysis of xylan involves the action of a number of different hydrolytic enzymes. The yeast Saccharomyces cerevisiae has been used extensively in traditional food and beverage processes (baking, brewing and winemaking), as well as for the production of ethanol (potable alcohol and fuel extenders) and single-cell protein (protein supplements in food and animal feed). S. cerevisiae therefore has complete GRAS (Generally Regarded as Safe) status. However, the yeast S. cerevisiae can neither degrade nor utilize complex polysaccharides, including xylan. Through recombinant DNA technology, S. cerevisiae can be complemented by heterologous polysaccharase-encoding genes, thereby broadening its substrate range and facilitating a direct bioconversion of polysaccharides to valuable commodities, such as potable ethanol, protein supplements and industrial enzymes. In this study, the successful expression and co-expression of a β xylanase gene (Trichoderma reesei xyn2) and two β xylosidase genes (Bacillus pumilus xynB and A. niger xlnD) in S. cerevisiae, is described. Expression of these genes was obtained with the aid of multi-copy episomal yeast plasmids pJC1, pDLG1, pDLG4 and pRLR1. These plasmids contain either the derepressible alcohol dehydrogenase 2 (ADH2) or the constitutive phosphoglycerate kinase 1 (PGK1) promoter and terminator sequences. The enhanced production of recombinant enzymes by S. cerevisiae in a rich medium, without the risk of losing the episomal vector, was obtained by disrupting the uracil phosphoribosyltransferase (FUR1) gene in the plasmid-containing S. cerevisiae strains. This step ensured auto-selection of the URA3-bearing expression plasmids in rich growth medium. High level expression of the T. reesei β xylanase gene in S. cerevisiae enabled the yeast to degrade xylan to short xylo-oligosaccharides, but very little monomeric D xylose was formed. Both β xylosidase genes enabled S. cerevisiae to degrade short xylo-oligosaccharides like xylobiose and xylotriose. Co-expression of the β xylanase and the B. pumilus β xylosidase led to a small increase in the β xylanase activity, but a substantial decrease in the amount of β xylosidase activity. This recombinant yeast strain was unable to degrade xylan to D xylose. Expression of the T. reesei β xylanase with the A. niger β xylosidase gene enabled this strain to completely degrade xylan to its monomeric constituents, D xylose. / AFRIKAANSE OPSOMMING: Hemisellulose, wat hoofsaaklik uit xilaan bestaan, is ná sellulose, die volopste hernubare polisakkaried in landbouafval. Xilaan is 'n komplekse polimeer wat bestaan uit 'n β-D-1,4-gekoppelde xilopiranoseruggraat wat in sommige gevalle ook sykettings bevat. Ensimatiese afbraak van xilaan benodig die werking van hele aantal hidrolitiese ensieme. Die gis Saccharomyces cerevisiae word al vir baie jare in die voedsel- en drankbedryf (bak van brood en die maak van bier en wyn), asook vir die produksie van etanol (vir menslik gebruik en as brandstof aanvuller) en enkelselproteïene (proteïenaanvulling vir mens en dier) gebruik en het daarom volledige GRAS (Generally Regarded As Safe) status. Ongelukkig kan S. cerevisiae nie komplekse polisakkariede, xilaan ingesluit, afbreek of as koolstofbron benut nie. Met behulp van rekombinante-DNA-tegnologie kan S. cerevisiae gekomplementeer word met die nodige gene wat kodeer vir polisakkariedafbrekende ensieme om sodoende die gis in staat te stel om 'n wyer verskeidenheid van substrate af te breek en te benut. Dit sal lei tot die direkte bio-omskakeling van polisakkariede na bruikbare produkte soos etanol, proteïenaanvullers en ensieme vir industriële gebruik. In hierdie proefskrif word die suksesvolle uitdrukking asook die gesamentlike uitdrukking van 'n xilanasegeen (Trichoderma reesei xyn2) en twee β-xilosidasegene (Bacillus pumilus xynB en A. niger xlnD) in S. cerevisiae beskryf. Multikopie episomale plasmiede pJC1, pDLG1, pDLG4 en pRLR1 met die glukose onderdrukbare alkoholdehidrogenase 2 (ADH2) of die konstitutiewe fosfogliseraatkinase 1 (PGK1)- promoter en -termineerder is vir hierdie doel gebruik. Verhoogde produksie van die rekombinante ensieme deur S. cerevisiae in 'n ryk medium, sonder dat die gis die episomale plasmiedvektore verloor is moontlik gemaak deur die urasielfosforibosieltransferasegeen (FUR1) van hierdie giste te onderbreek met behulp van die LEU2-geen. Op hierdie manier word daar outomaties vir giste wat die URA3-uitdrukkingsplasmiede bevat geselekteer, selfs in ryk medium. Hoë vlak uitdrukking van T. reesei se xilanasegeen het S. cerevisiae in staat gestel om xilaan tot kort xilo-oligosakkariede af te breek, maar byna geen monomeriese D-xilose is gevorm nie. Albei die β-xilosidasegene het die gis in staat gestel om kort xilo-oligosakkariede soos xilobiose en xilotriose na D-xilose af te breek. Die gesamentlike uitdrukking van die xilanasegeen en B. pumilus se β-xilosidase geen het 'n klein toename in die xilanase-aktiwiteit tot gevolg gehad, maar 'n drastiese afname in die β-xilosidase-aktiwiteit. Hierdie rekombinante ras kon dus nie xilaan tot xilose afbreek nie. Uitdrukking van T. reesei se β-xilanasegeen saam met die β-xilosidasegeen van A. niger, het S. cerevisiae in staat gestel om xilaan tot sy monomeriese boustene, D-xilose, af te breek.