Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast

The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc-) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc- S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in gluco...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2004, Vol.70 (1), p.159-166
Main Authors: Maris, A.J.A. van, Geertman, J.M.A, Vermeulen, A, Groothuizen, M.K, Winkler, A.A, Piper, M.D.W, Dijken, J.P. van, Pronk, J.T
Format: Article
Language:English
Subjects:
artificial selection
Biological and medical sciences
biosynthesis
cell culture
chemostat culture
continuous systems
Culture Media
Directed Molecular Evolution
Evolution
Fermentation
Fundamental and applied biological sciences. Psychology
gene expression
Gene Expression Regulation, Fungal
Glucose
Glucose - metabolism
glucose tolerance
hexose transporters
messenger RNA
microarray technology
Microbiology
Mutation
Physiology and Biotechnology
Proteome
pyruvate decarboxylase
Pyruvate Decarboxylase - genetics
Pyruvate Decarboxylase - metabolism
pyruvic acid
Pyruvic Acid - metabolism
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - growth & development
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Transcription, Genetic
transcriptome analysis
transporters
yeasts
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Summary:The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc-) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc- S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C2 compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc- strain without these growth defects. An acetate-independent Pdc- mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C2 independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc- S. cerevisiae mutant (TAM) that could grow in batch cultures (mu(max) = 0.20 h-1) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter-1 was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass-1 h-1 during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose-1.
ISSN:0099-2240
1098-5336
DOI:10.1128/AEM.70.1.159-166.2004