Abstract:
The production of ethanol from starch has been investigated in four genetically modified Saccharomyces cerevisiae strains (YPG/AB, YA7, YPG/MM and YPB-G). Three of the four strains produce the Aspergillus awamori glucoamylase together with either the Bacillus subttilis (YPG/AB, YA7) or the mouse (YPG/MM) á-amylase as separately secreted polypeptides. YPB-G, on the other hand, secretes a bifunctional fusion protein that contains both the B.subtilis á,amylase and the A.awamori glucoamylase activities. Substrate utilisation, biomass growth, ethanol production and plasmid stability were all studied in both starch and glucose-containing media in shake flasks and in a New Brunswick fermenter. Much higher growth rates were found any of the four strains was grown on glucose. YPG/AB showed the most efficient utilisation of starch for ethanol production with the lowest levels of reducing sugars accumulating in the medium. This strain produced 18.8 g/L and 43.8 g/L ethanol from 50 g/L and 100 g/L starch respectively. The superior performance of YPG/AB and YA7 as compared to YPB-G was found to correlate with its higher level of á -amylase activity in starch containing media. YPB-G, which secretes the bifunctional fusion protein, could produce 35.2 g/L ethanol in media with starch concentrations above 100 g/L, while YPG/MM did not produce ethanol from starch because of its negligible secretion of glucoamylase. Furthermore, a simple structured mathematical model was proposed to analyze the growth and enzyme secretion kinetics of the recombinant strain (YPB-G) in a complex medium containing glucose. The parameters obtained by the nonlinear estimation techniques were validated against experiments that were not used in the calibration of the models.
