While active site occupancy, allostery, and protein covalent modification enable fast metabolic responses, metabolism is also regulated on longer time scales by changes in enzyme concentrations. As bioengineering efforts typically involve sustained growth of genetically modified microbes, understanding the interplay of transcriptional and faster acting regulatory mechanisms in control of metabolism is especially industrially relevant. Two recent studies in yeast shed light on this topic, by probing how metabolism adapts when enzyme levels are dysregulated due to transcription factor knockout.
Fendt et al. measured transcript, enzyme, and metabolite concentrations in wild-type yeast and a GCR2
transcription factor mutant [35•
]. This mutant exhibits decreased glycolytic and enhanced TCA cycle enzyme expression. The resulting changes in enzyme levels are buffered by changes in substrate concentrations so as to maintain metabolic flux a property essential for cell growth. Interestingly, this observed anti-correlation (enzyme down, substrate up, and vice versa) was stronger for glycolytic and TCA cycle compounds than for reaction cofactors (e.g., NAD+
). This is in keeping with recent data from E. coli,
which suggests that cofactor sites are usually substrate-saturated, whereas sites for glycolytic and TCA metabolites often are not [3
]. Changing glycolytic and TCA metabolite levels therefore is the more straightforward road to modulating flux than varying cofactors. In follow-up experiments, expression of specific glycolytic enzymes was reduced using a Tet-repressible promoter system in place of the more globally perturbed GCR2
mutant. Consistent with flux compensation through greater substrate active-site occupancy, this brought about local accumulation of the enzyme substrates. This accommodation of altered enzyme levels by substrate concentration changes is in accordance with flux control being distributed over multiple pathway enzymes, as argued by metabolic control analysis [36
]. It is also possible, however, that flux control resides outside of glycolysis or the TCA cycle per se, e.g., in demand for ATP or in regulation of glucose uptake. Resolving such issues is of more than academic interest. For example, if glycolytic flux is limited by demand for ATP, then even over-expression of the full glycolytic pathway would not enhance production of the biofuel ethanol; however, expressing a general ATP hydrolyase might do so.
Amino acid biosynthesis in yeast provides such a case where flux control typically resides outside of a metabolic pathway. Except in cases (such as glycine) where a single amino acid can be made multiple different ways, steady-state pathway flux must match consumption by protein synthesis. Moxley et al. probed the means by which yeast lacking the GCN4
transcription factor maintain amino acid production when enzyme levels are inappropriately low [37•
]. An inhibitor of histidine biosynthesis was titrated into cultures of wild type and mutant yeast to induce matched growth rates. This inhibitor caused amino acid starvation and thus, in wild-type cells, GCN4
-induced expression of amino acid biosynthetic enzymes. In the absence of such expression, amino acid levels were lower, and this appeared to restore flux by relief of feedback inhibition by metabolite end products.
Glycine biosynthesis presented an interesting exception to the norm of metabolic flux conservation. In the absence of GCN4 activation, yeast synthesize glycine primarily from serine; upon its activation, glycine is instead made from threonine. While Moxley et al. found no overall correlation between transcript levels and biosynthetic fluxes, the GCN4-driven shift in the route of glycine biosynthesis was accompanied by matching changes in enzyme transcript levels.
These two studies examined different aspects of yeast metabolism under experimental conditions that demanded flux conservation. In the face of deranged transcription, yeast maintained flux through altered levels of metabolic substrates [35•
] and of allosteric effectors [37•
]. When an overt shift in pathway usage occurred (i.e., of routes of glycine biosynthesis), this was driven by transcriptional regulation. In a biotechnology setting, the flux maintenance would have been a depressing outcome a failure of metabolic engineering. In a more natural setting, rather than maintaining similar flux, the transcription-defective mutants would have been out-competed for scarce nutrients and ended up flux-deficient. Thus, while these studies highlight the potential for tradeoffs between metabolite and enzyme levels, further work is needed to understand how these tradeoffs play out in natural and industrial environments. To enable effective engineering, identifying points of flux control is particularly important. Figuring out the best way to do this via metabolomics and other modern methods is an open area of research.