Masters Degrees (Institute for Wine Biotechnology)

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Browsing Masters Degrees (Institute for Wine Biotechnology) by Author "Eksteen, Jeremy Michael"

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    Construction of recombinant Saccharomyces cerevisiae strains for starch utilisation
    (Stellenbosch : Stellenbosch University, 2002-12) Eksteen, Jeremy Michael; Pretorius, I. S.; Van Rensburg, P.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology .
    ENGLISH ABSTRACT: Starch-containing agricultural crops are widely available as feedstocks for the production of fuel ethanol, potable spirits or beer, single-cell protein (animal feed) and high-fructose corn syrups (sweeteners). Starch-rich crops, such as maize, rye, barley and wheat, are usually used for the production of whisky. One of the first steps in the production of whisky is to boil the raw starch at temperatures exceeding 100°C. This gelatinisation step is performed to disrupt and solubilise the starch granules to make them more accessible for enzymatic hydrolysis. After this cooking process, the starch is liquefied by a-amylase and then saccharified by glucoamylase and a debranching enzyme. Lipomyces kononenkoae and Saccharomycopsis fibuligera secrete highly effective a-amylases and glucoamylases, making them two of the most efficient raw-starchdegrading yeasts known. However, L. kononenkoae and S. fibuligera cannot be used in existing industrial fermentations because of their low ethanol tolerance, slow growth rate, catabolite repression, poorly characterised genetics and lack of GRAS (Generally Regarded As Safe) status. This study is divided into two sections. The aim of the first section was to clone a gene (LKA2) encoding a novel starch-degrading enzyme, a second a-amylase (Lka2p) from L. kononenkoae. LKA2 was cloned into a multicopy plasmid, the yeast episomal plasmid, YEp352, under the control of the phosphoglycerate kinase promoter (PGK1 p) and terminator (PGKh) expression cassette. This recombinant plasmid was designated pJUL3 and transformed into a laboratory strain of S. cerevisiae, I1278b. Plate and liquid assays revealed that the recombinant yeast secreted active a-amylase into the medium. The optimum pH for Lka2p was pH 3.5 and the optimum temperature 60°C. The aim of the second part of the study was to construct recombinant strains of S. cerevisiae secreting a-amylase and/or glucoamylase. The individual genes were cloned into a yeast-integrating plasmid, Ylp5, under the control of the PGK1p-PGK1.,-expression cassette. Two indigenous yeasts were selected on the basis of their ability to utilise raw starch, L. kononenkoae and S. fibuligera, as gene donors. Eight constructs containing the L. kononenkoae a-amylase genes, LKA 1 and LKA2, and the S. fibuligera a-amylase (SFA 1) and glucoamylase (SFG1) genes were prepared: four single-cassette plasmids expressing the individual coding sequences under the control of the PGK1 p-PGK1.,- expression cassette, resulting in plPLKA 1, pIPLKA2, plPSFA 1 and pIPSFG1, respectively; two double-cassette plasm ids (expressing both LKA 1 and LKA2 under the control of the PGK1p-PGK1 .,-expression cassette, and SFA 1 and SFG1 under their respective native promoters and terminators), resulting in pIPLKA1/2 and pIPSFAG, respectively, and two single-cassette plasmids expressing SFA 1 and SFG1 with their native promoters and terminators, resulting in pSFA 1 and pSFG1, respectively. The respective constructs were transformed into a laboratory strain of S. cere visiae , L1278b. By homologous recombination, each plasmid was integrated into the yeast genome at the ura3 locus. S. cerevisiae L:1278b that had been transformed with plPLKA 1/2, LKA 1 and LKA2 under the control of the PGK1 rrPGK1,expression cassette resulted in the highest levels of a-amylase activity when assayed for amylolytic activity in a liquid medium. This recombinant strain resulted in the most efficient starch utilisation in batch fermentations, consuming 80% of starch and producing 6 gIL of ethanol after 156 hours of fermentation. The strain expressing SFG1 under the control of the PGK1rrPGK1,expression cassette gave the highest levels of glucoamylase activity.' These results confirmed that co-expression of a-amylase and/or glucoamylase synergistically enhance starch degradation. This study paves the way for the development of efficient starch-degrading strains of S. cerevisiae for the production of whisky, beer and biofuel ethanol.