Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Subject "Amylases"

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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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    Expression of novel amylases in Saccharomyces cerevisiae for the efficient conversion of raw starch to bioethanol
    (Stellenbosch : Stellenbosch University, 2017-03) Cripwell, Rosemary Anne; Van Zyl, Willem Heber; Rose, Shuanita; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Starchy biomass is an ideal, abundant substrate for bioethanol production. The cost effective conversion of starch requires a fermenting yeast that is able to produce starch hydrolysing enzymes and ferment glucose to ethanol in one step called consolidated bioprocessing (CBP). Despite the advantages, CBP yeasts have not yet been employed for the industrial processing of raw starch during bioethanol production. Molecular biology has enabled the optimised expression of synthetically produced genes in Saccharomyces cerevisiae. The Aspergillus tubingensis raw starch hydrolysing α-amylase (amyA) and glucoamylase (glaA) encoding genes were codon optimised using different strategies and expressed in S. cerevisiae Y294. However, compared to the native coding sequences for the amyA and glaA genes, adapted synonymous codon usage resulted in a decrease in extracellular enzyme activity of 72% (30 nkat.ml-1) and 69% (4 nkat.ml-1), respectively. Additional fungal amylase encoding genes (native and codon optimised) were expressed in S. cerevisiae Y294 and then screened for starch hydrolysis. Subsequently, S. cerevisiae Y294 laboratory strains were constructed to co-express the best α-amylase and glucoamylase gene variants and evaluated for raw starch fermentation. During raw starch fermentations, the S. cerevisiae Y294[TemG_Opt-TemA_Nat] strain displayed the highest carbon conversion (based on the percentage starch converted on a mol carbon basis) of 85%, compared to 54% displayed by the S. cerevisiae Y294[AmyA-GlaA] benchmark strain. Therefore, the native α-amylase (temA_Nat) and codon optimised glucoamylase (temG_Opt) genes, both originating from Talaromyces emersonii, presented the best amylase combination and were selected for further evaluation. Amylolytic S. cerevisiae Ethanol Red™ and M2n industrial strains were constructed using the amdS marker (encoding for acetamidase). Strains co-expressing the temA_Nat and temG_Opt genes were selected for growth on acetamide as the sole nitrogen source. Amylolytic S. cerevisiae strains (Ethanol Red T12 and M2n T1) were compared in a CBP process (20% raw corn starch) at 30°C and 37°C. The maximum ethanol concentration produced at 30°C by the S. cerevisiae Ethanol Red T12 and M2n T1 strains was 86.5 g.l-1 and 99.4 g.l-1, respectively. Fermentations were supplemented with different dosages of STARGEN 002™, an exogenous GSHE (granular starch hydrolysing enzyme) cocktail, to compare the amylolytic yeast strains to an industrial simultaneous saccharification and fermentation (SSF) process. Fermentation results for the S. cerevisiae Ethanol Red T12 strain with 10% of the recommended STARGEN™ dosage compared well with the SSF using S. cerevisiae Ethanol Red™ containing the full recommended STARGEN™ dosage, both having carbon conversions of 50% after 48 hours and 93% after 192 hours. This study also highlights the application of novel industrial amylolytic yeasts in combination with STARGEN™ for decreased fermentations times. At 37°C, the amylolytic S. cerevisiae Ethanol Red T12 strain performed better than the S. cerevisiae M2n T1 strain, demonstrating its potential as a drop-in CBP yeast for existing bioethanol plants that use cold hydrolysis processes. The study also provided a novel enzyme combination (TemA_Nat and TemG_Opt) that efficiently hydrolyses raw corn starch. Finally, new light was shed on the importance of synonymous codon usage and the expression of native genes versus their codon optimised variants.