Doctoral Degrees (Microbiology)

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Browsing Doctoral Degrees (Microbiology) by Author "Cripwell, Rosemary Anne"

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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.