The growth kinetic characterisation of Saccharomyces cerevisiae strains transformed with amylase genes

English: The direct fermentation of starch to ethanol using an amylase-producing yeast is of interest as an alternative to the conventional fermentation processes, which utilise commercial amylases. Starch is an abundant renewable biopolymer, comprising two major components, namely amylose (α-I,4-linked D-glucose residues) and amylopectin (α-I,4 and α-I,6-linked D-glucose residues), typically constituting 80 % of cereal starches. Efficient starch degradation, therefore, necessitates the use of α- and glucoamylases, together with an α-I,6 debranching activity. Naturally amylolytic yeasts are not suited to fermentations, whereas Saccharomyces cerevisiae, known for its strong fermentation capacity, lacks amylolytic activity (with the exception of S. cerevisiae var. diastaticus, which has weak glucoamylase activity). Consequently, the diploid "Sigma" strain of S. cerevisiae was transformed with different combinations of amylase genes from Lipomyces spencermartinsiae (with the PGK] promoter), and Saccharomycopsis fibuligera (with its natural promoters) using an integrating plasmid in a single copy form. These recombinant strains, provided by the University of Stellenbosch, were evaluated in respect of their ability to ferment starch to ethanol. Recombinant strains of S. cerevisiae containing the S. fibuligera amylase genes with non-integrating plasmids in a multi-copy and a single copy form, provided by the University of the Free State, were also evaluated. Notable differences in the hydrolysis zones on starch agar plates indicated that the type of starch medium used and the amylase produced exerted a significant effect. The dimensions of these hydrolysis zones were a poor indicator of the performance of the strains in liquid starch media. Anoxic cultivations in shake flasks and in 2-1 bioreactors containing a 2 % starch medium yielded less than 109 ethanol.l-1 over a 200 h incubation period. Aerobic growth yielded more biomass and, therefore, higher amylase values and higher rates of starch hydrolysis, but with no detectable amounts of ethanol. Initial evaluations indicated poor amylase activity, particularly with strains containing the S. fibuligera amylase genes. The limited amylase production by a strain containing the S. fibuligera amylase genes was not due to proteolytic activity or intracellular enzyme . accumulation. Strains containing the L. spencermartinsiae amylase genes gave the best overall results. However, a strain containing a combination of both a-amylase and . glucoamylase yielded disappointing results. The curious multi-phasic growth profile obtained with this strain, accompanied by a delay in amylase production, suggested regulation of the PGKl promoter by glucose. However, Northern blot analyses indicated very low levels of glucoamylase mRNA, with no clear indication of induction of the PGKl promoter. A recombinant strain (strain steIl7) with both glucoamylase genes (LKAll from L. spencermartinsiae and GLUI from S. fibuligera) proved to be the most prormsmg. Further evaluation in 15-1 bioreactors resulted in the production of ea. 21 g ethanol.l-1 from 55 g starch.l-1 by strain stell7. Despite a relatively high α.-amylase activity of 500 U.l-1 after 150 h, the slow rate of enzyme production remained the rate-limiting step. Although some of these recombinant strains were capable of complete starch hydrolysis, the slow rate of starch saccharification and the concomitant low ethanol productivity rendered these strains unattractive for commercial application.