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13 C metabolic flux analysis ( 13 C MFA) is an important systems biology technique that has been used to investigate microbial metabolism for decades. The heterotrimer Snf1 kinase complex plays a key role in the preference  Saccharomyces cerevisiae  exhibits for glucose over galactose, a phenomenon known as glucose repression or carbon catabolite repression. The  SIP1  gene, encoding a part of this complex, has received little attention, presumably, because its knockout lacks a growth phenotype. We present a fluxomic investigation of the relative effects of the presence of galactose in classically glucose-repressing media and/or knockout of  SIP1  using a multi-scale variant of  13 C MFA known as 2-Scale  13 C metabolic flux analysis (2S- 13 C MFA). In this study, all strains have the galactose metabolism deactivated ( gal1 Δ background) so as to be able to separate the metabolic effects purely related to glucose repression from those arising from galactose metabolism. The resulting flux profiles reveal that the presence of galactose in classically glucose-repressing conditions, for a CEN.PK113-7D  gal1 Δ background, results in a substantial decrease in pentose phosphate pathway (PPP) flux and increased flow from cytosolic pyruvate and malate through the mitochondria toward cytosolic branched-chain amino acid biosynthesis. These fluxomic redistributions are accompanied by a higher maximum specific growth rate, both seemingly in violation of glucose repression. Deletion of  SIP1  in the CEN.PK113-7D  gal1 Δ cells grown in mixed glucose/galactose medium results in a further increase. Knockout of this gene in cells grown in glucose-only medium results in no change in growth rate and a corresponding decrease in glucose and ethanol exchange fluxes and flux through pathways involved in aspartate/threonine biosynthesis. Glucose repression appears to be violated at a 1/10 ratio of galactose-to-glucose. Based on the scientific literature, we may have conducted our experiments near a critical sugar ratio that is known to allow galactose to enter the cell. Additionally, we report a number of fluxomic changes associated with these growth rate increases and unexpected flux profile redistributions resulting from deletion of  SIP1  in glucose-only medium.

Flux-Enabled Exploration of the Role of Sip1 in Galactose Yeast Metabolism