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Appl Environ Microbiol, June 1998, p. 2133-2140, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Effects of Pyruvate Decarboxylase Overproduction on Flux Distribution at the Pyruvate Branch Point in Saccharomyces cerevisiae

Pim van Hoek,1 Marcel T. Flikweert,1 Quirina J. M. van der Aart,2 H. Yde Steensma,1,2 Johannes P. van Dijken,1 and Jack T. Pronk1,*

Department of Microbiology, Kluyver Institute of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft,1 and Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, 2333 AL Leiden,2 The Netherlands

Received 2 January 1998/Accepted 15 March 1998

A multicopy plasmid carrying the PDC1 gene (encoding pyruvate decarboxylase; Pdc) was introduced in Saccharomyces cerevisiae CEN.PK113-5D. The physiology of the resulting prototrophic strain was compared with that of the isogenic prototrophic strain CEN.PK113-7D and an empty-vector reference strain. In glucose-grown shake-flask cultures, the introduction of the PDC1 plasmid caused a threefold increase in the Pdc level. In aerobic glucose-limited chemostat cultures growing at a dilution rate of 0.10 h-1, Pdc levels in the overproducing strain were 14-fold higher than those in the reference strains. Levels of glycolytic enzymes decreased by ca. 15%, probably due to dilution by the overproduced Pdc protein. In chemostat cultures, the extent of Pdc overproduction decreased with increasing dilution rate. The high degree of overproduction of Pdc at low dilution rates did not affect the biomass yield. The dilution rate at which aerobic fermentation set in decreased from 0.30 h-1 in the reference strains to 0.23 h-1 in the Pdc-overproducing strain. In the latter strain, the specific respiration rate reached a maximum above the dilution rate at which aerobic fermentation first occurred. This result indicates that a limited respiratory capacity was not responsible for the onset of aerobic fermentation in the Pdc-overproducing strain. Rather, the results indicate that Pdc overproduction affected flux distribution at the pyruvate branch point by influencing competition for pyruvate between Pdc and the mitochondrial pyruvate dehydrogenase complex. In respiratory cultures (dilution rate, <0.23 h-1), Pdc overproduction did not affect the maximum glycolytic capacity, as determined in anaerobic glucose-pulse experiments.

* Corresponding author. Mailing address: Kluyver Institute of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. Phone: 31 15 2783214. Fax: 31 15 2782355. E-mail: j.t.pronk{at}stm.tudelft.nl.

Appl Environ Microbiol, June 1998, p. 2133-2140, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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