Prolonged
chemostat cultivation of Saccharomyces cerevisiae under glucose
limitation leads to a partial loss of fermentative capacity.
Mickel Jansen (1), Adham Hassane (1), Jack Pronk (1), Han de Winde (2)
(1) Kluyver Laboratory of Biotechnology, Delft University of Technology,
Julianalaan 67, 2628 BC Delft, The Netherlands; (2) DSM Life Sciences, Bakery
Ingredients Division, Technology Cluster, PO Box 1, 2600 MA Delft, The
Netherlands (j.h.dewinde@tnw.tudelft.nl)
Prolonged
glucose-limited chemostat cultivation of Saccharomyces cerevisiae resulted in a
significant decrease in fermentative capacity (FC). After 200 generations
(dilution rate of 0.1 h-1) a five-fold decrease in FC was observed.
The residual glucose concentration decreased from 20 to 8 mg/l, indicating the
selection of cells with higher uptake affinity for glucose. Genome-wide
transcript analysis did not reveal major alterations in gene expression.
Contrastingly, the in vitro activity of most glycolytic enzymes, especially in
the lower part of glycolysis, decreased significantly. Additionally, clear
changes in cellular morphology were observed. To test whether the changes
resulted from physiological adaptation or mutations, new chemostats were
inoculated with single-cell isolates from the prolonged chemostat cultivations.
The new cultures exhibited identical phenotypes and physiological parameters.
Hence, changes occurring in the prolonged chemostats were caused by mutative
selection. Further characterization of the selected mutant cells revealed a
decrease in mu-max (0.35 h-1 instead of 0.42 h-1 in
wild-type) and a delayed ethanol production after glucose-pulsing. This
'Crabtree-negative behavior' lasted for two hours, after which ethanol
production finally occurred. We conclude that increased culture age in
chemostats has a major effect on baker's yeast metabolism, leading to
progressive loss of FC and the inducton of a Crabtree-negative phenotype.