Engineering of Promoter Replacement Cassettes for Fine-Tuning of Gene Expression in Saccharomyces cerevisiae

ABSTRACT The strong overexpression or complete deletion of a gene gives only limited information about its control over a certain phenotype or pathway. Gene function studies based on these methods are therefore incomplete. To effect facile manipulation of gene expression across a full continuum of possible expression levels, we recently created a library of mutant promoters. Here, we provide the detailed characterization of our yeast promoter collection comprising 11 mutants of the strong constitutive Saccharomyces cerevisiae TEF1 promoter. The activities of the mutant promoters range between about 8% and 120% of the activity of the unmutated TEF1 promoter. The differences in reporter gene expression in the 11 mutants were independent of the carbon source used, and real-time PCR confirmed that these differences were due to varying levels of transcription (i.e., caused by varying promoter strengths). In addition to a CEN/ARS plasmid-based promoter collection, we also created promoter replacement cassettes. They enable genomic integration of our mutant promoter collection upstream of any given yeast gene, allowing detailed genotype-phenotype characterizations. To illustrate the utility of the method, the GPD1 promoter of S. cerevisiae was replaced by five TEF1 promoter mutants of different strengths, which allowed analysis of the impact of glycerol 3-phosphate dehydrogenase activity on the glycerol yield.

[1]  E. Nevoigt,et al.  Genetic engineering of brewing yeast to reduce the content of ethanol in beer. , 2002, FEMS yeast research.

[2]  P. Philippsen,et al.  New heterologous modules for classical or PCR‐based gene disruptions in Saccharomyces cerevisiae , 1994, Yeast.

[3]  J. Broach,et al.  UASrpg can function as a heterochromatin boundary element in yeast. , 1999, Genes & development.

[4]  Peter Ruhdal Jensen,et al.  Modulation of Gene Expression Made Easy , 2002, Applied and Environmental Microbiology.

[5]  Yong-Su Jin,et al.  Optimal Growth and Ethanol Production from Xylose by Recombinant Saccharomyces cerevisiae Require Moderate d-Xylulokinase Activity , 2003, Applied and Environmental Microbiology.

[6]  J. Hegemann,et al.  A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. , 2002, Nucleic acids research.

[7]  Thomas Fiedler,et al.  A new efficient gene disruption cassette for repeated use in budding yeast , 1996, Nucleic Acids Res..

[8]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[9]  Johann M. Rohwer,et al.  Metabolic Control Analysis of Glycerol Synthesis in Saccharomyces cerevisiae , 2002, Applied and Environmental Microbiology.

[10]  G. Kunze,et al.  A wide-range integrative yeast expression vector system based on Arxula adeninivorans-derived elements , 2004, Journal of Industrial Microbiology and Biotechnology.

[11]  Gregory Stephanopoulos,et al.  Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression. , 2003, Metabolic Engineering.

[12]  P. Philippsen,et al.  Identification of two genes coding for the translation elongation factor EF‐1 alpha of S. cerevisiae. , 1984, The EMBO journal.

[13]  G. Lidén,et al.  Physiological response to anaerobicity of glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae , 1997, Applied and environmental microbiology.

[14]  G. Stephanopoulos,et al.  Tuning genetic control through promoter engineering. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[15]  R. Niedenthal,et al.  Vector systems for heterologous expression of proteins in Saccharomyces cerevisiae. , 2002, Methods in enzymology.

[16]  E. Nevoigt,et al.  Reduced pyruvate decarboxylase and increased glycerol‐3‐phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae , 1996, Yeast.

[17]  R. Müller,et al.  Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. , 1994, Nucleic acids research.

[18]  J M Thevelein,et al.  GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway , 1994, Molecular and cellular biology.

[19]  Y. Kawai,et al.  Utilization of the TEF1-a gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae , 1998, Applied Microbiology and Biotechnology.

[20]  J. Keasling,et al.  Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria. , 2000, Metabolic engineering.

[21]  E. Craig,et al.  The Glycine-Phenylalanine-Rich Region Determines the Specificity of the Yeast Hsp40 Sis1 , 1999, Molecular and Cellular Biology.

[22]  C. Raymond,et al.  General method for plasmid construction using homologous recombination. , 1999, BioTechniques.

[23]  P. R. Jensen,et al.  Artificial promoters for metabolic optimization. , 1998, Biotechnology and bioengineering.

[24]  P. Philippsen,et al.  Sequence and promoter analysis of the highly expressed TEF gene of the filamentous fungus Ashbya gossypii , 1994, Molecular and General Genetics MGG.

[25]  Friedrich Srienc,et al.  Flow cytometry as a useful tool for process development: rapid evaluation of expression systems. , 2002, Journal of biotechnology.

[26]  P. Barré,et al.  Modulation of Glycerol and Ethanol Yields During Alcoholic Fermentation in Saccharomyces cerevisiae Strains Overexpressed or Disrupted for GPD1 Encoding Glycerol 3‐Phosphate Dehydrogenase , 1997, Yeast.

[27]  E. Nevoigt,et al.  Engineering of Saccharomyces cerevisiae for the production of L-glycerol 3-phosphate. , 2004, Metabolic engineering.

[28]  P. Barré,et al.  Glycerol Overproduction by Engineered Saccharomyces cerevisiae Wine Yeast Strains Leads to Substantial Changes in By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase , 1999, Applied and Environmental Microbiology.

[29]  K. Hammer,et al.  The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters , 1998, Applied and Environmental Microbiology.

[30]  P. R. Jensen,et al.  The level of glucose‐6‐phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains , 2003, Yeast.

[31]  L. Tao,et al.  Directed evolution of copy number of a broad host range plasmid for metabolic engineering. , 2005, Metabolic engineering.

[32]  K. Thorn,et al.  Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae , 2004, Yeast.

[33]  A. Voegler,et al.  Reduction of BiP Levels Decreases Heterologous Protein Secretion in Saccharomyces cerevisiae(*) , 1996, The Journal of Biological Chemistry.

[34]  S. Dequin,et al.  Glycerol export and glycerol-3-phosphate dehydrogenase, but not glycerol phosphatase, are rate limiting for glycerol production in Saccharomyces cerevisiae. , 2001, Metabolic engineering.