Browsing by Author "Sakwa, Liana-Lisa"

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    Cloning and expression of fungal alpha-amylase genes in Saccharomyces cerevisiae with integrated glucoamylase gene for raw starch conversion into bioethanol
    (Stellenbosch : Stellenbosch University, 2017-03) Sakwa, Liana-Lisa; Rose, Shuanita; Viljoen-Bloom, M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Increasing population numbers and the rapid growth of technology and industry have resulted in an increase in energy demand. Biomass-based fuels (biofuels) have received considerable interest as an alternative transport fuel as biomass is abundant, cheap and renewable. Starch is a good feedstock for bioethanol production with a mature technology established in the USA. However, the current starch-to-ethanol conversion process requires a high energy input and high amylolytic enzyme loadings for liquefaction, resulting in economic challenges. The yeast Saccharomyces cerevisiae is traditionally the preferred host for bioethanol production due to its high ethanol productivity, tolerance and high fermentation capacity, but is unable to utilise or ferment starch. Genetic engineering allows the construction of amylolytic S. cerevisiae strains that can convert starch to glucose and ferment the latter to ethanol. The application of raw starch hydrolysing enzymes could reduce the process time and cost of ethanol production, thus improving its economic feasibility. In this study, a literature and database search was conducted to obtain DNA sequences of genes encoding raw starch hydrolysing amylases. The Aureobasidium pullulans ApuA, Aspergillus terreus AteA, Cryptococcus sp. S-2 CryA and Saccharomycopsis fibuligera SfiA α-amylase encoding genes were synthesised and expressed on an episomal multicopy vector in a S. cerevisiae laboratory strain using the Trichoderma reesei xyn2 secretion signal. The S. cerevisiae Y294[AteA] and Y294[ApuA] strains displayed the highest levels of volumetric activity for the recombinant α-amylases (3.20 U.ml-1 and 2.57 U.ml-1, respectively) when grown in SC–URA medium. The recombinant AteA and ApuA proteins were glycosylated and displayed pH optima between pH 4 and 5. Both enzymes were stable at 30°C and maintained up to 80% activity after 5 days. The ApuA and AteA genes were co-expressed with the Aspergillus tubingensis GlaA glucoamylase to generate the S. cerevisiae Y294[ApuA-GlaA] and Y294[AteA-GlaA] strains, respectively. When cultivated on 200 g.l-1 raw starch, the Y294[AteA-GlaA] strain produced 43.81 g.l-1 ethanol after 192 hours, which was significantly higher than the Y294[AmyA-GlaA] benchmark strain (41.02 g.l-1) and the Y294[ApuA-GlaA] strain (32.83 g.l-1). The Y294[AteA-GlaA] strain displayed a maximum yield of 57 g.l-1 ethanol in fermentations supplemented with STARGENTM 002 (commercial enzyme cocktail), indicating the margin of improvement possible in improving process efficiency. Assessment of the Y294[AteA-GlaA] strain using various optimisation strategies concluded that additional glucoamylase would improve the fermentation rate and thus decrease the required fermentation time. High substrate loading reduced the fermentation efficiency, with up to a 50% improvement in starch conversion when the substrate loading was halved. In this study, ethanol production was strain dependent (as only one parental strain was used), signifying that any further increase in enzyme production will not result in an increased ethanol yield, but will instead result in an improved fermentation rate. This study provides insights into the dynamics of hydrolysis of raw starch in a single-step Consolidated Bioprocessing (CBP) process. The importance of using appropriate enzyme ratios is highlighted as it ensures the improved efficiency and effectiveness of a CBP system. The knowledge obtained from this study is useful in the realisation of economic benefits of process integration in CBP for commercial starch–based biofuel production streams.