Comparative Proteome Analysis of Saccharomyces cerevisiae Grown in Chemostat Cultures Limited for Glucose or Ethanol

Abstract

The use of chemostat culturing enables investigation of steady-state physiological characteristics and adaptations to nutrient-limited growth, while all other relevant growth conditions are kept constant. We examined and compared the proteomic response of wild-type Saccharomyces cerevisiae CEN.PK113-7D to growth in aerobic chemostat cultures limited for carbon sources being either glucose or ethanol. To obtain a global overview of changes in the proteome, we performed triplicate analyses using two-dimensional gel electrophoresis and identified proteins of interest using MS. Relative quantities of about 400 proteins were obtained and analyzed statistically to determine which protein steady-state expression levels changed significantly under glucose-or ethanollimited conditions. Interestingly, only enzymes involved in central carbon metabolism showed a significant change in steady-state expression, whereas expression was only detected in one of both carbon source-limiting conditions for 15 of these enzymes. Side effects that were previously reported for batch cultivation conditions, such as responses to continuous variation of specific growth rate, to carbon-catabolite repression, and to accumulation of toxic substrates, were not observed. Moreover, by comparing our proteome data with corresponding mRNA data, we were able to unravel which processes in the central carbon metabolism were regulated at the level of the proteome, and which processes at the level of transcriptome. Importantly, we show here that the combined approach of chemostat cultivation and comprehensive proteome analysis allowed us to study the primary effect of single limiting conditions on the yeast proteome. Molecular & Cellular Proteomics 4:1-11, 2005. Two-dimensional (2D)1 gel electrophoresis is a powerful tool to visualize hundreds of proteins at a time, which in combination with MS leads to their identification. This technology has been applied to the yeast Saccharomyces cerevisiae for the large-scale identification of more than 400 proteins, resulting in yeast reference maps (1-7). Other S. cerevisiae studies used 2D gel electrophoresis to obtain an overview of global changes in the yeast proteome as function of stimuli such as cadmium (8), lithium (9), H 2 O 2 (10), or sorbic acid (11).Most of these differential proteome studies on S. cerevisiae were performed on cells cultured in batch mode, i.e. in shake flasks or in reactors. Batch cultivation makes use of a closed system, in which all nutrients are in excess at the start of the cultivation. In terms of microbial physiology, such batch cultivation is relatively poorly controlled, because the composition of the growth medium and consequently the growth rate changes continuously. The yeast cells take up nutrients from the media while metabolites are excreted in the culture system, and their growth arrests when one of the nutrients is depleted or when too many toxic substrates are accumulated. The high concentration of carbon source in the culture, which is essential for this type of cultu...

Comparative Proteomic Analysis of Tolerance and Adaptation of EthanologenicSaccharomyces cerevisiaeto Furfural, a Lignocellulosic Inhibitory Compound

“…Since it has been reported that the central carbon metabolism of S. cerevisiae is controlled to a large extent via posttranscriptional mechanisms in chemostat studies (11,32,33), quantitative RT-PCR was not carried out to substantiate our findings concerning the involvement of the central carbon metabolism in cellular response to the presence of furfural. Instead, the levels of intracellular NADH, NAD ϩ , NADH/ NAD ϩ , and ATP/ADP were measured by LC/MS (Fig.…”

Development and application of proteomics technologies in Saccharomyces cerevisiae

“…This study revealed that metabolic fluxes in the acetate and glycerol pathway were significantly different in cells growing on xylose compared with those growing on glucose. Kolkman et al studied yeast grown in chemostat cultures limited for glucose and ethanol [44], which enabled the differential analysis of protein expression levels under glycolytic and gluconeogenic conditions. By using multiple 2D gels (Figure 1c,d) it was possible to perform a decent statistical analysis to determine significant changes in protein expression levels.…”

Transcriptomic Analysis Identifies Growth Rate Modulation as a Component of the Adaptation of Mycobacteria to Survival inside the Macrophage

“…Data obtained in a microarray analysis of organisms grown in a chemostat have been shown to be more reproducible and accurate than batch culture DNA array data (6,12,13,17). A combination of a chemostat culture and DNA microarrays was used previously to study the effects of oxygen limitation on the gene expression profile of M. tuberculosis (2).…”