Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by browse.metadata.advisor "Cordero Otero, Ricardo R."

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    Development of improved α-amylases
    (Stellenbosch : University of Stellenbosch, 2005-03) Ramachandran, Nivetha; Cordero Otero, Ricardo R.; Pretorius, Isak S.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
    The technological advancement of modern human civilisation has, until recently, depended on extensive exploitation of fossil fuels, such as oil, coal and gas, as sources of energy. Over the last few decades, greater efforts have been made to economise on the use of these nonrenewable energy resources, and to reduce the environmental pollution caused by their consumption. In a quest for new sources of energy that will be compatible with a more sustainable world economy, increased emphasis has been place on researching and developing alternative sources of energy that are renewable and safer for the environment. Fuel ethanol, which has a higher octane rating than gasoline, makes up approximately two-thirds of the world’s total annual ethanol production. Uncertainty surrounding the longterm sustainability of fuel ethanol as an energy source has prompted consideration for the use of bioethanol (ethanol from biomass) as an energy source. Factors compromising the continued availability of fuel ethanol as an energy source include the inevitable exhaustion of the world’s fossil oil resources, a possible interruption in oil supply caused by political interference, the superior net performance of biofuel ethanol in comparison to gasoline, and a significant reduction in pollution levels. It is to be expected that the demand for inexpensive, renewable substrates and cost-effective ethanol production processes will become increasingly urgent. Plant biomass (including so-called ‘energy crops’, agricultural surplus products, and waste material) is the only foreseeable sustainable source of fuel ethanol because it is relatively low in cost and in plentiful supply. The principal impediment to more widespread utilisation of this important resource is the general absence of low cost technology for overcoming the difficulties of degrading the recalcitrant polysaccharides in plant biomass to fermentable sugars from ethanol can be produced. A promising strategy for dealing with this obstacle involves the genetic modification of Saccharomyces cerevisiae yeast strains for use in an integrated process, known as direct microbial conversion (DMC) or consolidated bioprocessing (CBP). This integrated process differs from the earlier strategies of SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation, in which enzymes from external sources are used) in that the production of polysaccharide-degrading enzymes, the hydrolysis of biomass and the fermentation of the resulting sugars to ethanol all take place in a single process by means of a polysaccharidefermenting yeast strain. The CBP strategy offers a substantial reduction in cost if S. cerevisiae strains can be developed that possess the required combination of substrate utilisation and product formation properties. S. cerevisiae strains with the ability to efficiently utilise polysaccharides such as starch for the production of high ethanol yields have not been described to date. However, significant progress towards the development of such amylolytic strains has been made over the past decade. With the aim of developing an efficient starch-degrading, high ethanol-yielding yeast strain, our laboratory has expressed a wide variety of heterologous amylase-encoding genes in S. cerevisiae. This study forms part of a large research programme aimed at improving these amylolytic ‘prototype’ strains of S. cerevisiae. More specifically, this study investigated the LKA1- and LKA2-encoded α-amylases (Lka1p and Lka2p) from the yeast Lipomyces kononenkoae. These α-amylases belong to the family of glycosyl hydrolases (EC 3.2.1.1) and are considered to be two of the most efficient raw-starch-degrading enzymes. Lka1p functions primarily on the α-1,4 linkages of starch, but is also active on the α-1,6 linkages. In addition, it is capable of degrading pullulan. Lka2p acts on the α-1,4 linkages. The purpose of this study was two-fold. The first goal was to characterise the molecular structure of Lka1p and Lka2p in order to better understand the structure-function relationships and role of specific amino acids in protein function with the aim of improving their substrate specificity in raw starch hydrolysis. The second aim was to determine the effect of yeast cell flocculence on the efficiency of starch fermentation, the possible development of high-flocculating, LKA1-expressing S. cerevisiae strains as ‘whole-cell biocatalysts’, and the production of high yields of ethanol from raw starch. In order to understand the structure-function relationships in Lka1p and Lka2p, standard computational and bioinformatics techniques were used to analyse the primary structure. On the basis of the primary structure and the prediction of the secondary structure, an N-terminal region (1-132 amino acids) was identified in Lka1p, the truncation of which led to the loss of raw starch adsorption and also rendered the protein less thermostable. Lka1p and Lka2p share a similar catalytic TIM barrel, consisting of four highly conserved regions previously observed in other α-amylase members. Furthermore, the unique Q414 of Lka1p located in the catalytic domain in place of the invariant H296 (TAKA amylase), which offers transition state stabilisation in α-amylases, was found to be involved in the substrate specificity of Lka1p. Mutational analysis of Q414 performed in the current study provides a basis for understanding the various properties of Lka1p in relation to the structural differences observed in this molecule. Knowing which molecular features of Lka1p contribute to its biochemical properties provides us with the potential to expand the substrate specificity properties of this α-amylase towards more effective processing of its starch and related substrates. In attempting to develop ‘whole-cell biocatalysts’, the yeast’s capacity for flocculation was used to improve raw starch hydrolysis by S. cerevisiae expressing LKA1. It was evident that the flocculent cells exhibited physicochemical properties that led to a better interaction with the starch matrix. This, in turn, led to a decrease in the time interval for interaction between the enzyme and the substrate, thus facilitating faster substrate degradation in flocculent cells. The use of flocculation serves as a promising strategy to best exploit the expression of LKA1 in S. cerevisiae for raw starch hydrolysis. This thesis describes the approaches taken to investigate the molecular features involved in the function of the L. kononenkoae α-amylases, and to improve their properties for the efficient hydrolysis of raw starch. This study contributes to the development of amylolytic S. cerevisiae strains for their potential use in single-step, cost-effective production of fuel ethanol from inexpensive starch-rich materials.
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    Enhancing xylose utilisation during fermentation by engineering recombinant Saccharomyces cerevisiae strains
    (Stellenbosch : Stellenbosch University, 2007-12) Thanvanthri Gururajan, Vasudevan; Cordero Otero, Ricardo R.; Pretorius, Isak S.; Van Rensburg, Pierre; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Xylose is the second most abundant sugar present in plant biomass. Plant biomass is the only potential renewable and sustainable source of energy available to mankind at present, especially in the production of transportation fuels. Transportation fuels such as gasoline can be blended with or completely replaced by ethanol produced exclusively from plant biomass, known as bio-ethanol. Bio-ethanol has the potential to reduce carbon emissions and also the dependence on foreign oil (mostly from the Middle East and Africa) for many countries. Bio-ethanol can be produced from both starch and cellulose present in plants, even though cellulosic ethanol has been suggested to be the more feasible option. Lignocellulose can be broken down to cellulose and hemicellulose by the hydrolytic action of acids or enzymes, which can, in turn, be broken down to monosaccharides such as hexoses and pentoses. These simple sugars can then be fermented to ethanol by microorganisms. Among the innumerable microorganisms present in nature, the yeast Saccharomyces cerevisiae is the most efficient ethanol producer on an industrial scale. Its unique ability to efficiently synthesise and tolerate alcohol has made it the ‘workhorse’ of the alcohol industry. Although S. cerevisiae has arguably a relatively wide substrate utilisation range, it cannot assimilate pentose sugars such as xylose and arabinose. Since xylose constitutes at least one-third of the sugars present in lignocellulose, the ethanol yield from fermentation using S. cerevisiae would be inefficient due to the non-utilisation of this sugar. Thus, several attempts towards xylose fermentation by S. cerevisiae have been made. Through molecular cloning methods, xylose pathway genes from the natural xylose-utilising yeast Pichia stipitis and an anaerobic fungus, Piromyces, have been cloned and expressed separately in various S. cerevisiae strains. However, recombinant S. cerevisiae strains expressing P. stipitis genes encoding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) had poor growth on xylose and fermented this pentose sugar to xylitol. The main focus of this study was to improve xylose utilisation by a recombinant S. cerevisiae expressing the P. stipitis XYL1 and XYL2 genes under anaerobic fermentation conditions. This has been approached at three different levels: (i) by creating constitutive carbon catabolite repression mutants in the recombinant S. cerevisiae background so that a glucose-like environment is mimicked for the yeast cells during xylose fermentation; (ii) by isolating and cloning a novel xylose reductase gene from the natural xylose-degrading fungus Neurospora crassa through functional complementation in S. cerevisiae; and (iii) by random mutagenesis of a recombinant XYL1 and XYL2 expressing S. cerevisiae strain to create haploid xylose-fermenting mutant that showed an altered product profile after anaerobic xylose fermentation. From the data obtained, it has been shown that it is possible to improve the anaerobic xylose utilisation of recombinant S. cerevisiae to varying degrees using the strategies followed, although ethanol formation appears to be a highly regulated process in the cell. In summary, this work exposits three different methods of improving xylose utilisation under anaerobic conditions through manipulations at the molecular level and metabolic level. The novel S. cerevisiae strains developed and described in this study show improved xylose utilisation. These strains, in turn, could be developed further to encompass other polysaccharide degradation properties to be used in the so-called consolidated bioprocess.
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    Genetic engineering of Saccharomyces cerevisiae for efficient polysaccharide utilisation
    (Stellenbosch : University of Stellenbosch, 2005-03) Gundllapalli, Sarath Babu; Cordero Otero, Ricardo R.; Pretroius, Isak S.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
    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.