Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions

• Published in: Microbiology (Society for General Microbiology) 2001-02, Vol.147 (2), p.391-401
• Main Authors: Pearce, Amanda K, Crimmins, Kay, Groussac, Evelyne, Hewlins, Michael J. E, Dickinson, J. Richard, Francois, Jean, Booth, Ian R, Brown, Alistair J. P
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Fermentation; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Fungal; Glycolysis; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Magnetic Resonance Spectroscopy; Microbiology; Mission oriented research; Mutation; Mycology; Phosphofructokinase-1 - genetics; Phosphofructokinase-1 - metabolism; Physiology and metabolism; Pyruvate Kinase - genetics; Pyruvate Kinase - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - physiology; Transcription, Genetic - genetics
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-147-2-391

Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK 1 Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504 UR-INRA 792, Département de Génie Biochimique et Alimentaire, Institut National des Sciences Appliquées, 31077 Toulouse Cedex 04, France 2 Department of Chemistry, Cardiff University, PO Box 912, Cardiff CF10 3TB, UK 3 Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK 4 Author for correspondence: Alistair J. P. Brown. Tel: [phone]. Fax: [phone]. e-mail: al.brown{at}abdn.ac.uk Yeast phosphofructo-1-kinase (Pf1k) and pyruvate kinase (Pyk1) are allosterically regulated enzymes that catalyse essentially irreversible reactions in glycolysis. Both the synthesis and activity of these enzymes are tightly regulated. To separate experimentally the control of Pf1k and Pyk1 synthesis from their allosteric regulation, a congenic set of PFK1 , PFK2 and PYK1 mutants was constructed in which these wild-type coding regions were driven by alternative promoters. Mutants carrying PGK1 promoter fusions displayed normal rates of growth, glucose consumption and ethanol production, indicating that the relatively tight regulation of Pyk1 and Pf1k synthesis is not essential for glycolytic control under fermentative growth conditions. Mutants carrying fusions to an enhancer-less version of the PGK1 promoter ( PGK1 767 ) expressed Pyk1 and Pf1k at about 2·5-fold lower levels than normal. Physiological and metabolic analysis of the PFK1 PFK2 double mutant indicated that decreased Pf1k had no significant effect on growth, apparently due to compensatory increases in its positive effector, fructose 2,6-bisphosphate. In contrast, growth rate and glycolytic flux were reduced in the PGK1 767 –PYK1 mutant, which had decreased Pyk1 levels. Unexpectedly, the reduced Pyk1 levels caused the flow of carbon to the TCA cycle to increase, even under fermentative growth conditions. Therefore, Pyk1 exerts a significant level of control over both the rate and direction of carbon flux in yeast. Keywords: Glycolysis, metabolic flux, pyruvate kinase, phosphofructokinase, yeast physiology Abbreviations: MCA, metabolic control analysis; Pf1k, 6-phosphofructo-1-kinase; Pyk1, pyruvate kinase a Present address: MRC Radiation and Genome Stability Unit, Harwell, Didcot, Oxfordshire OX11 ORD, UK. b Present address: Quest International, Menstrie, Clackmannanshire SK11 7ES, UK.