Conference Dates

March 8-13, 2009


Ethanol can be used as a complete fuel or as an octane enhancer, and has the advantages of being renewable and environmentally friendly. Ethanol produced by a fermentation process, generally referred to as bioethanol, is considered to be a partial solution to the worldwide energy crisis. Traditionally, industrial bioethanol fermentation involves two major steps: starch hydrolysis and fermentation. Since the key microorganism, Saccharomyces cerevisiae, lacks amylolytic activity and is unable to directly utilize starch for proliferation and fermentation, it requires intensive amount of energy and pure starch hydrolyzing enzymes to gelatinize, liquefy and dextrinize the raw starch before fermentation.

It has been suggested that genetically engineered yeast which expresses amylolytic enzymes could potentially perform simultaneous starch hydrolysis and fermentation. This improvement could greatly reduce the capital and energy costs in current bioethanol producing plants and make bioethanol production more economical. In this project, a novel yeast strain of Saccharomyces cerevisiae was genetically engineered in such a way that barley alpha-amylase was constitutively expressed and immobilized on the yeast cell surface. This particular alpha-amylase was selected based on its superior kinetic properties and its pH optimum which is compatible with the pH of yeast culture media. The cDNA encoding barley alpha-amylase, with a secretion signal sequence, was fused to the cDNA encoding the C-terminal half of a cell wall anchoring protein, alpha-agglutinin. The fusion gene was cloned downstream of a constitutive promoter ADH1 in a yeast episomal plasmid pAMY. The constructed plasmid, pAMY, also contains ampicillin and blasticidin resistance genes for selection of the plasmid harbouring clones in E. coli and yeast, respectively. By developing a starch plate assay, in which clear haloes formed around the successful colonies on a YPD-starch agar plate after staining with iodine vapour, it was shown pAMY harbouring yeast demonstrated detectable amylolytic activity. In addition, cell suspensions of pAMY harbouring yeast were incubated with 1% soluble starch in 16 mM sodium acetate buffer, pH 4.5, 45°C. The majority of soluble starch was converted into maltose within 6 hours and alpha-amylase activity was detected only in the cell pellet fraction and not in the culture supernatant. In batch fermentation studies using 2% soluble wheat starch as sole carbon source, even though pAMY harbouring yeast was able to hydrolyse 75% of soluble starch in 160 hours under the fermentation conditions, no ethanol was produced. This was felt to be due to insufficient alpha-amylase activity which resulted from the enzyme being anchored on the cell wall by alpha-agglutinin. Also, plasmid stability of pAMY harbouring yeast without the presence of selecting antibiotics was studied during batch fermentation experiments. The results suggest that soluble starch may positively select plasmid harbouring yeast cells to stabilize the population of plasmid harbouring cells over plasmid free cells in the fermentor during prolonged batch fermentation. Further research using alternative cell surface anchoring systems are being planned to produce yeast with high ethanol yields for industrial applications.