Masters Degrees (Microbiology)

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Browsing Masters Degrees (Microbiology) by Subject "Amylolysis"

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    Recombinant Saccharomyces cerevisiae strains for improved bioethanol production from starch-based substrates
    (Stellenbosch : Stellenbosch University, 2020-03) Myburgh, Marthinus Wessel; Viljoen-Bloom, Marinda; Rose, Shuanita; Cripwell, Rosemary Anne; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Increased concern regarding climate change has highlighted the need for alternative sources of energy and fuel. The production of bioethanol from starch is a mature technology and is in the favourable position to replace fossil fuels, particularly in the transport sector. However, certain bottlenecks associated with the starch-to-ethanol process increase the cost and have made it difficult for bioethanol to compete with fossil fuel prices. Major bottlenecks include the high cost of the substrate, the need for expensive exogenous enzyme cocktails and the energy requirements for heating and temperature regulation. These bottlenecks could be addressed through the use of cheaper starch-based substrates and the development of yeast strains for a single-step hydrolysis-and-fermentation approach called consolidated bioprocessing (CBP). CBP requires recombinant yeast strains to produce amylases that hydrolyse raw starch while simultaneously converting the sugar products to high ethanol titres. A number of amylolytic strains have been developed, but continued research strives to increase the levels of amylase production. One approach is to use stronger promoters since they are the main regulators of gene expression and essential for effective heterologous expression in yeast. In addition, advances in molecular techniques have made promoter engineering a viable approach to further increase the expression of heterologous genes. Two industrial amylolytic yeast strains, ER T12 and M2n T1, expressing both the Talaromyces emersonii α-amylase and glucoamylase genes, were evaluated for their ability to hydrolyse starch in untreated broken rice and ferment the sugars to ethanol. Cell-free hydrolysis trials showed that crude enzymes from the ER T12 strain hydrolysed broken rice with a similar saccharification yield as a commercial amylase cocktail. Both strains were able to convert starch to ethanol in a single step, reaching theoretical maximum ethanol titers (roughly 100 g/l) and converting 100% of the estimated available carbon to products. Complete replacement of exogenous enzyme addition was achieved with the ER T12 strain without reducing ethanol productivity or yield. The ER T12 strain therefore has full CBP capability on broken rice and is a particularly good contender for application in commercial ethanol plants. It is known that the most effective promoter-gene combination is required for optimal heterologous gene expression. Eight endogenous Saccharomyces cerevisiae promoters were investigated for expression of the Aspergillus terreus α-amylase gene in the S. cerevisiae Y294 laboratory strain. The RPS25AP, HXT7P, TPI1P and TEF1P promoters showed higher expression levels than the ENO1P benchmark promoter. In an effort to further increase amylase expression, six promoters were engineered to contain the S. cerevisiae RPS25Ai intron. The inclusion of the intron resulted in an overall increase in amylase activity, protein production and transcript levels. The top-performing Y294[DC-TEF] and Y294[DC-TDHi] strains delivered high ethanol titres (94% and 97% of the theoretical maximum, respectively) and outperformed the benchmark strain on raw corn starch and broken rice. This study showed that industrial amylolytic yeasts could be employed for the production of bioethanol from untreated broken rice. However, there is also scope to further improve existing amylolytic strains by selecting and engineering strong promoters for heterologous gene expression.