Unraveling condition-dependent networks of transcription factors that control metabolic pathway activity in yeast

Which transcription factors control the distribution of metabolic fluxes under a given condition? We address this question by systematically quantifying metabolic fluxes in 119 transcription factor deletion mutants of Saccharomyces cerevisiae under five growth conditions. While most knockouts did not affect fluxes, we identified 42 condition‐dependent interactions that were mediated by a total of 23 transcription factors that control almost exclusively the cellular decision between respiration and fermentation. This relatively sparse, condition‐specific network of active metabolic control contrasts with the much larger gene regulation network inferred from expression and DNA binding data. Based on protein and transcript analyses in key mutants, we identified three enzymes in the tricarboxylic acid cycle as the key targets of this transcriptional control. For the transcription factor Gcn4, we demonstrate that this control is mediated through the PKA and Snf1 signaling cascade. The discrepancy between flux response predictions, based on the known regulatory network architecture and our functional 13 C‐data, demonstrates the importance of identifying and quantifying the extent to which regulatory effectors alter cellular functions. Synopsis Effective control and modulation of cellular behavior is of paramount importance in medicine (Kreeger and Lauffenburger, 2010 ) and biotechnology (Haynes and Silver, 2009 ), and requires profound understanding of control mechanisms. In this study, we aim to elucidate the extent to which transcription factors control the operation of yeast metabolism. As a quantitative readout of metabolic function, we monitored the traffic of small molecules through various pathways of central metabolism by 13 C‐flux analysis (Sauer, 2006 ). The choosen growth conditions represent two different regulatory states of reduced (galactose) and maximal carbon source repression (glucose), as well as a different nitrogen metabolism and two common, permanent stress conditions. Depending on the growth condition, between 7 and 13% of the deleted transcription factors altered the determined flux ratios (Figure 3 ). Of the six quantified flux ratios, only the glycolysis/pentose phosphate pathway split, and the convergent ratio of anaplerosis and TCA cycle were controlled by the deleted transcription factors. Thus, we concluded that 23 transcription factors control flux distributions under at least one of the tested growth conditions, leading to 42 cond