Steady‐state and transient‐state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate‐decarboxylase activity

Flikweert, Marcel T.; Kuyper, Marko; Maris, Antonius J. A. van; Kötter, Peter; Dijken, Johannes P. van; Pronk, Jack T.

doi:10.1002/(sici)1097-0290(1999)66:1<42::aid-bit4>3.3.co;2-c

Cited by 17 publications

(32 citation statements)

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“…First, similar to a strain with reduced expression of PDC (11), the engineered strain exhibited a reduced maximum specific growth rate of 0.20 h Ϫ1 in glucoselimited chemostat cultures compared to 0.38 h Ϫ1 of the wild type. Second, like other strains of S. cerevisiae with reduced or zero PDC activity (11,13,35), it produced substantial amounts of pyruvate during exposure to glucose excess (Fig. 2).…”

Section: Discussionmentioning

confidence: 79%

Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures ofSaccharomyces cerevisiae

Maris

Luttik

Winkler³

et al. 2003

Appl Environ Microbiol

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Pyruvate decarboxylase-negative (Pdc ؊ ) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C 2 requirement of Pdc ؊ S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of L-carnitine to growth media did not restore growth of a Pdc ؊ strain on glucose, indicating that the C 2 requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc ؊ strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc ؊ GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc ؊ strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.

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Section: Discussionmentioning

confidence: 79%

Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures ofSaccharomyces cerevisiae

Maris

Luttik

Winkler³

et al. 2003

Appl Environ Microbiol

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“…Reductive metabolism sets in after transition of sugarlimited conditions to sugar excess due to a transient saturation of the flux capacity of the oxidative catabolism, causing overflow of the glycolytic flux at the level of pyruvate [1]. The transient adaptation of oxidative catabolism to the new extracellular conditions has relaxation times of minutes up to a few hours [8] and is usually provoked by pulse additions to chemostat cultivations [9,10,11]. Shift-up experiments inside of the oxidative region were also performed, by which the saturation and adaptation of the oxidative catabolism were quantified by a transient uncoupling of oxidative and reductive metabolism [12].…”

Section: Introductionmentioning

confidence: 99%

A small metabolic flux model to identify transient metabolic regulations in Saccharomyces cerevisiae

Herwig

Stockar

2002

Bioprocess and Biosystems Engineering

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The understanding of dynamic metabolic regulations is important for physiological studies and strain characterization tasks. The present study combined transient experiments with online metabolic flux analysis (MFA) in order to quantify metabolic regulations, namely carbon catabolite repression of respiration and transient acetic-acid production, in Saccharomyces cerevisiae during aerobic growth on glucose. The aim was to investigate which additional information can be gained from using a small metabolic flux model to study transient growth provoked by shift-up and shift-down experiments, compared to online monitoring alone. The MFA model allowed us to propose new correlations between pathways of the central metabolism. A linear correlation between glycolytic flux and respiratory capacity holds for shift-down and shift-up experiments. This confirmed that respiratory functions were subjected to carbon catabolite repression and suggested that respiratory capacity is controlled by the glycolytic flux rather than the glucose influx. Furthermore, the model showed that control of repression of respiration by the glycolytic flux was a dynamic phenomenon. Co-factor balancing within the MFA model showed that transient acetic-acid production indicated a transient limitation in another part of the central metabolism but not in oxidative phosphorylation. However, at super-critical growth rates and when coupling of anabolism and catabolism is resumed, the limitation shifts to oxidative phosphorylation, with the consequence that ethanol is formed. The online application of small metabolic flux models to transient experiments enhanced the physiological insight into transient growth and opens up the use of transient experiments as an efficient tool to understand dynamic metabolic regulations.

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“…Pyruvate decarboxylases convert pyruvate to acetaldehyde, a crucial step in the formation of ethanol. Deletion of PDC2 has previously been shown to result in a 3-to 4-fold reduction of Pdc activity in aerobic glucose-limited chemostat cultures, and in substantial production of pyruvate during batch cultivation on glucose (10). In the present study, PDC2 was deleted in strain IMK157, yielding strain IMK299.…”

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Anaplerotic Role for Cytosolic Malic Enzyme in Engineered Saccharomyces cerevisiae Strains

Zelle

Harrison²,

Pronk

et al. 2011

Appl Environ Microbiol

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Malic enzyme catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO 2 . The Saccharomyces cerevisiae MAE1 gene encodes a mitochondrial malic enzyme whose proposed physiological roles are related to the oxidative, malate-decarboxylating reaction. Hitherto, the inability of pyruvate carboxylasenegative (Pyc ؊ ) S. cerevisiae strains to grow on glucose suggested that Mae1p cannot act as a pyruvatecarboxylating, anaplerotic enzyme. In this study, relocation of malic enzyme to the cytosol and creation of thermodynamically favorable conditions for pyruvate carboxylation by metabolic engineering, process design, and adaptive evolution, enabled malic enzyme to act as the sole anaplerotic enzyme in S. cerevisiae. The Escherichia coli NADH-dependent sfcA malic enzyme was expressed in a Pyc ؊ S. cerevisiae background. When PDC2, a transcriptional regulator of pyruvate decarboxylase genes, was deleted to increase intracellular pyruvate levels and cells were grown under a CO 2 atmosphere to favor carboxylation, adaptive evolution yielded a strain that grew on glucose (specific growth rate, 0.06 ؎ 0.01 h ؊1 ). Growth of the evolved strain was enabled by a single point mutation (Asp336Gly) that switched the cofactor preference of E. coli malic enzyme from NADH to NADPH. Consistently, cytosolic relocalization of the native Mae1p, which can use both NADH and NADPH, in a pyc1,2⌬ pdc2⌬ strain grown under a CO 2 atmosphere, also enabled slow-growth on glucose. Although growth rates of these strains are still low, the higher ATP efficiency of carboxylation via malic enzyme, compared to the pyruvate carboxylase pathway, may contribute to metabolic engineering of S. cerevisiae for anaerobic, high-yield C 4 -dicarboxylic acid production.

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Steady‐state and transient‐state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate‐decarboxylase activity

Cited by 17 publications

References 0 publications

Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures ofSaccharomyces cerevisiae

Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures ofSaccharomyces cerevisiae

A small metabolic flux model to identify transient metabolic regulations in Saccharomyces cerevisiae

Anaplerotic Role for Cytosolic Malic Enzyme in Engineered Saccharomyces cerevisiae Strains

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