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The yeast Saccharomyces cerevisiae is unique among eukaryotes in exhibiting fast growth in both the presence and the complete absence of oxygen. Genome-wide transcriptional adaptation to aerobiosis and anaerobiosis was studied in assays using DNA microarrays. This technique was combined with chemostat cultivation, which allows controlled variation of a single growth parameter under defined conditions and at a fixed specific growth rate. Of the 6,171 open reading frames investigated, 5,738 (93%) yielded detectable transcript levels under either aerobic or anaerobic conditions; 140 genes showed a >3-fold-higher transcription level under anaerobic conditions. Under aerobic conditions, transcript levels of 219 genes were >3-fold higher than under anaerobic conditions.

Evolutionary engineering, which uses laboratory evolution to select for industrially relevant traits, is a popular strategy in the development of high-performing yeast strains for industrial production of fuels and chemicals. By integrating whole-genome sequencing, bioinformatics, classical genetics and genome-editing techniques, evolutionary engineering has also become a powerful approach for identification and reverse engineering of molecular mechanisms that underlie industrially relevant traits. New techniques enable acceleration of in vivo mutation rates, both across yeast genomes and at specific loci. Recent studies indicate that phenotypic trade-offs, which are often observed after evolution under constant conditions, can be mitigated by using dynamic cultivation regimes. Advances in research on synthetic regulatory circuits offer exciting possibilities to extend the applicability of evolutionary engineering to products of yeasts whose synthesis requires a net input of cellular energy.

Saccharomyces cerevisiae T23C ( p d a l : :TnSb/e) is an isogenic gene replacement mutant o f the wild-type strain S. cerevisiae T23D. The mutation causes a complete loss of pyruvate dehydrogenase activity. Pyruvate metabolism in this pyruvate-dehydrogenase-negative (Pdh -) strain was investigated in aerobic glucose-limited chemostat cultures, grown a t a dilution rate o f 0.10 h-l, and compared with the metabolism in the isogenic wild-type strain. Under these conditions, growth of the Pdh-strain was fully respiratory. Enzyme activities in cell-free extracts indicated that the enzymes pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A (acetyl-CoA) synthetase could provide a functional bypass o f the pyruvate dehydrogenase complex. Since this metabolic sequence involves ATP hydrolysis in the acetyl-CoA synthetase reaction, a negative effect of the p d a l : :TnSble mutation on the growth efficiency was anticipated. Indeed, the biomass yield o f the Pdh-strain [044 g biomass (g g l u~o s e ) -~] was significantly lower than that o f wild-type 5. cerevisiae [ Om52 g biomass (g g l u~o s e ) -~] .The effect o f the mutation o n biomass yield could be quantitatively explained in terms of a lower ATP yield from glucose catabolism and an increased ATP requirement for the synthesis of acetyl-CoA used in anabolism. Control experiments showed that the p d a l : :TnSble mutation did not affect biomass yield in ethanol-limited chemostat cultures. The results support the view that, during aerobic glucoselimited growth o f S. cerevisiae at l o w growth rates, the pyruvate dehydrogenase complex accounts for the major part of the pyruvate flux. Moreover, it is concluded that hydrolysis of pyrophosphate formed in the acetyl-CoA synthetase reaction does not contribute significantly to energy transduction in t h i s yeast. Respiratory-def icient cells did n o t contribute t o glucose metabolism in the chemostat cultures and were probably formed upon plating. (Wenzel et al., 1992). This mutation confers resistance to the antibiotic phleomycin and completely abolishes pyruvate dehydrogenase activity (Wenzel e t al., 1992). Both strains were stored as frozen Physiology and energetics of Pdh-S. cerevisiae KeywordsThe mineral medium, supplemented with vitamins, was prepared according to Verduyn e t al. (1992). Vitamins were filtersterilized and added after heat sterilization of the mineral medium. Glucose was sterilized separately at 110 "C. The concentration of ethanol or glucose in the reservoir medium was 5 g 1-'. Cultures were checked for the occurrence of oscillations (Parelukar e t al., 1986;Sonnleitner, 1991) by continuous registration of the dissolved-oxygen concentration and CO, production. All data presented refer to steady states without detectable oscillations.Control of culture purity and homogeneity. The purity of chemostat cultures was routinely checked by phase contrast microscopy at 1000 x magnification, and by plating of culture samples on YPD agar plates. Stability of the strain carrying the p...

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