Cellobiose consumption uncouples extracellular glucose sensing and glucose metabolism inSaccharomyces cerevisiae

Abstract

Glycolysis is central to energy metabolism in most organisms and is highly regulated to enable optimal growth. In the yeast Saccharomyces cerevisiae, feedback mechanisms that control flux through glycolysis span transcriptional control to metabolite levels in the cell. Using a cellobiose consumption pathway, we decoupled glucose sensing from carbon utilization, revealing new modular layers of control that induce ATP consumption to drive rapid carbon fermentation. Alterations of the beta subunit of phosphofructokinase-1 (PFK2), H ϩ -plasma membrane ATPase (PMA1), and glucose sensors (SNF3 and RGT2) revealed the importance of coupling extracellular glucose sensing to manage ATP levels in the cell. Controlling the upper bound of cellular ATP levels may be a general mechanism used to regulate energy levels in cells, via a regulatory network that can be uncoupled from ATP concentrations under perceived starvation conditions. IMPORTANCE Living cells are fine-tuned through evolution to thrive in their native environments. Genome alterations to create organisms for specific biotechnological applications may result in unexpected and undesired phenotypes. We used a minimal synthetic biological system in the yeast Saccharomyces cerevisiae as a platform to reveal novel connections between carbon sensing, starvation conditions, and energy homeostasis.KEYWORDS PMA1, cellobiose, glucose sensors, metabolomics M ost microorganisms favor glucose as their primary carbon source, as reflected in their genetic programs hard-wired for this preference. Central to carbon metabolism is glycolysis, which is finely tuned to the dynamic state of the cell due to the fact that glycolysis first consumes ATP before generating additional ATP equivalents. To avoid catastrophic depletion of ATP, the yeast Saccharomyces cerevisiae has evolved a transient ATP hydrolysis futile cycle coupled to gluconeogenesis (1). Glycolysis in yeast is also tightly coupled to glucose transport into the cell, entailing three extracellular glucose-sensing mechanisms and at least one intracellular glucose signaling pathway (2).Synthetic biology and metabolic engineering of yeast hold promise to convert this microorganism into a "cell factory" to produce a wide range of chemicals derived from renewable resources or those unattainable through traditional chemical routes. However, many applications require tapping into metabolites involved in central carbon metabolism, a daunting challenge as living cells have numerous layers of feedback regulation that fine-tune growth to changing environments. Cellular regulation evolved

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“…3). Furthermore, a recent study revealed that the plasma membrane ATPase (PMA1) of cellobiose-fermenting S. cerevisiae is in a carbon starvation-like state [60]. Although we unable to exactly pinpoint the cause of the reduced TaBgl1 glycosylation in cellobiose-containing medium, we propose that carbon starvation-like state may be at least in part responsible for that.…”

Neither 1G nor 2G fuel ethanol: setting the ground for a sugarcane-based biorefinery using an iSUCCELL yeast platform

“…Transcriptional and metabolite profiling have revealed that yeast cells fermenting cellobiose are subjected to severe physiological changes, compared to cells fermenting glucose, as reflected in the activation of mitochondrial function and a decrease in amino acid biosynthesis, and in a carbon starvation-like state of the plasma membrane ATPase (Pma1) (Lin et al 2014;Chomvong et al 2017). Furthermore, when cultivated in cellobiose medium, yeast cells accumulate high levels of trehalose and of intermediate metabolites in the γ-aminobutyrate (GABA) shunt pathway, improving the strain's tolerance to oxidative stress (Kim et al 2014b;Yun et al 2018).…”