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

[摘要] English: The direct fermentation of starch to ethanol using an amylase-producing yeast is ofinterest as an alternative to the conventional fermentation processes, which utilisecommercial amylases. Starch is an abundant renewable biopolymer, comprising twomajor 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 cerealstarches. Efficient starch degradation, therefore, necessitates the use of α- andglucoamylases, together with an α-I,6 debranching activity. Naturally amylolytic yeastsare not suited to fermentations, whereas Saccharomyces cerevisiae, known for its strongfermentation capacity, lacks amylolytic activity (with the exception of S. cerevisiae var.diastaticus, which has weak glucoamylase activity). Consequently, the diploid Sigmastrain of S. cerevisiae was transformed with different combinations of amylase genesfrom Lipomyces spencermartinsiae (with the PGK] promoter), and Saccharomycopsisfibuligera (with its natural promoters) using an integrating plasmid in a single copy form.These recombinant strains, provided by the University of Stellenbosch, were evaluated inrespect of their ability to ferment starch to ethanol. Recombinant strains of S. cerevisiaecontaining the S. fibuligera amylase genes with non-integrating plasmids in a multi-copyand 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. Thedimensions of these hydrolysis zones were a poor indicator of the performance of thestrains in liquid starch media. Anoxic cultivations in shake flasks and in 2-1 bioreactorscontaining a 2 % starch medium yielded less than 109 ethanol.l-1 over a 200 h incubationperiod. Aerobic growth yielded more biomass and, therefore, higher amylase values andhigher rates of starch hydrolysis, but with no detectable amounts of ethanol. Initialevaluations indicated poor amylase activity, particularly with strains containing theS. fibuligera amylase genes. The limited amylase production by a strain containing theS. fibuligera amylase genes was not due to proteolytic activity or intracellular enzyme. accumulation. Strains containing the L. spencermartinsiae amylase genes gave the bestoverall results. However, a strain containing a combination of both a-amylase and. glucoamylase yielded disappointing results. The curious multi-phasic growth profileobtained with this strain, accompanied by a delay in amylase production, suggestedregulation of the PGKl promoter by glucose. However, Northern blot analyses indicatedvery low levels of glucoamylase mRNA, with no clear indication of induction of thePGKl promoter. A recombinant strain (strain steIl7) with both glucoamylase genes(LKAll from L. spencermartinsiae and GLUI from S. fibuligera) proved to be the mostprormsmg. Further evaluation in 15-1 bioreactors resulted in the production ofea. 21 g ethanol.l-1 from 55 g starch.l-1 by strain stell7. Despite a relatively highα.-amylase activity of 500 U.l-1 after 150 h, the slow rate of enzyme production remainedthe rate-limiting step. Although some of these recombinant strains were capable ofcomplete starch hydrolysis, the slow rate of starch saccharification and the concomitantlow ethanol productivity rendered these strains unattractive for commercial application.

[发布机构] University of the Free State