In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae. I. Experimental observations

• Published in: Biotechnology and bioengineering 1997-07, Vol.55 (2), p.305-316
• Main Authors: Theobald, U, Mailinger, W, Baltes, M, Rizzi, M, Reuss, M
• Format: Article
• Language: English
• Subjects: adenine nucleotide pool; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; glucose effect; glycolysis; intracellular metabolites; metabolic dynamics; Methods. Procedures. Technologies; Microbial engineering. Fermentation and microbial culture technology; plant biochemistry; plant physiology; Saccharomyces cerevisiae
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(19970720)55:2<305::AID-BIT8>3.0.CO;2-M

The goal of this work was to obtain rapid sampling technique to measure transient metabolites in vivo. First, a pulse of glucose was added to a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s intervals and quenched using methods that depend on the metabolite measured. Extracellular glucose, excreted products, as well as glycolytic intermediates (G6P, F6P, FBP, GAP, 3-PG, PEP, Pyr) and cometabolites (ATP, ADP, AMP, NAD+, NADH) were measured using enzymatic or HPLC methods. Significant differences between the adenine nucleotide concentrations in the cytoplasm and mitochondria indicated the importance of compartmentation for the regulation of the glycolysis. Changes in the intra- and extracellular levels of metabolites confirmed that glycolysis is regulated on a time scale of seconds.