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Redesigning biology towards specific purposes requires a functional understanding of genetic circuits. We present a quantitative in-depth study on the regulation of the methanol-specific MOX promoter system (PMOX) at the single-cell level. We investigated PMOX regulation in the methylotrophic yeast Hansenula (Ogataea) polymorpha with respect to glucose-mediated carbon catabolite repression. This promoter system is particularly delicate as the glucose as carbon and energy source in turn represses MOX promoter activity. Decoupling single cells from population activity revealed a hitherto underrated ultrasensitivity of the MOX promoter to glucose repression. Environmental control with single-cell technologies enabled quantitative insights into the balance between activation and repression of PMOX with respect to extracellular glucose concentrations. While population-based studies suggested full MOX promoter derepression at extracellular glucose concentrations of ∼1 g L(-1), we showed that glucose-mediated catabolite repression already occurs at concentrations as low as 5 × 10(-4) g L(-1) These findings demonstrate the importance of uncoupling single cells from populations for understanding the mechanisms of promoter regulation in a quantitative manner.

TheMOXpromoter inHansenula polymorphais ultrasensitive to glucose-mediated carbon catabolite repression