Gene Regulation in Continuous Cultures: A Unified Theory for Bacteria and Yeasts

2019

Preprint

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The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose.This repression has been attributed to CRP-mediated transcriptional inhibition and EIIA Glcmediated inducer exclusion. The growing evidence against the first mechanism has led to the postulate that the repression is driven by inducer exclusion. The literature shows that in fully induced cells, inducer exclusion reduces the permease activity only 2-fold. However, it is conceivable that inducer exclusion drastically reduces the permease activity in partially induced cells. We measured the decline of lactose permease activity due to inducer exclusion in partially induced cells, but found that the permease activity decreased no more than 6-fold.We show that the repression is small because these experiments are performed in the presence of chloramphenicol. Indeed, when glucose is added to a culture growing on glycerol and TMG, but no chloramphenicol, lac is repressed 900-fold, of which inducer exclusion accounts for only 3-fold repression The remainder is due to reversal of the positive feedback loop, i.e., the decline of the intracellular TMG level leads to a lower permease level, which reduces the intracellular TMG level even further. The repression in the absence of chloramphenicol is therefore primarily due to positive feedback, which does not exist during measurements of inducer exclusion.

Section: Comparison Of Our Results To Studies In the Literaturementioning

confidence: 99%

Deconstructing glucose-mediated catabolite repression of thelacoperon ofEscherichia coli: I. Inducer exclusion, by itself, cannot account for the repression

Aggarwal

2019

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“…When the Mut + strain is grown in batch cultures of methanol + glycerol and methanol + sorbitol, there is diauxic growth, but methanol is the unpreferred substrate during growth on methanol + glycerol, and the preferred substrate during growth on methanol + sorbitol (Ramón et al ., 2007). Such mixtures, which display diauxic growth in batch cultures, exhibit a characteristic substrate concentration profile in continuous cultures (Egli et al ., 1986; Noel and Narang, 2009) (Supplementary Fig. S1a).…”

Section: Resultsmentioning

confidence: 99%

“…During single-substrate growth, the specific substrate consumption rate usually increases linearly with D up to washout (Pirt, 1965), but during mixed-substrate growth, the specific substrate consumption rates increase linearly with D only in the dual-limited regime (Egli et al ., 1986; Noel and Narang, 2009) (Supplementary Fig. S1b).…”

Section: Resultsmentioning

confidence: 99%

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P_AOX1expression in mixed-substrate continuous cultures ofKomagataella phaffii(Pichia pastoris) is completely determined by methanol consumption regardless of the secondary carbon source

Singh

2021

Preprint

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The expression of recombinant proteins by the AOX1 promoter of Komagataella phaffii is typically induced by adding methanol to the cultivation medium. Since growth on methanol imposes a high oxygen demand, the medium is often supplemented with an additional "secondary" carbon source which serves to reduce the consumption of methanol, and hence, oxygen. Early research recommended the use of glycerol as the secondary carbon source, but more recent studies recommend the use of sorbitol because glycerol represses PAOX1 expression. To assess the validity of this recommendation, we measured the steady state concentrations of biomass, residual methanol, and AOX1 over a wide range of dilution rates (0.02-0.20 h-1) in continuous cultures of the Mut+ strain fed with methanol, methanol + glycerol, and methanol + sorbitol. We find that when the specific AOX1 expression and methanol uptake rates for each of the three feeds are plotted against each other, they collapse into a single hyperbolic curve. The specific AOX1 expression rate is therefore completely determined by the specific methanol uptake rate regardless of the existence (present/absent) and type (repressing/non-repressing) of the secondary carbon source. In particular, cultures fed with methanol + glycerol and methanol + sorbitol that consume methanol at equal rates also express the protein at equal rates and levels. Now, it turns out that the simple unstructured model developed by Egli and co-workers can predict the specific methanol uptake rates of single- and mixed-substrate cultures over a wide range of dilution rates and feed concentrations. By combining this model with our data, we derive simple formulas that predicts the protein expression rates and levels of single- and mixed-substrate cultures over a wide range of conditions.

“…The entry of substrates such as glucose that are transported by the phosphotransferase system (PTS) results in the formation of dephosphorylated enzyme IIA glc , which binds to lactose permease and other non-PTS transport enzymes, thus inhibiting entry of the inducer into the cell. (b) Positive feedback (Narang 1998;Narang and Pilyugin 2007;Noel and Narang 2009; fi gure 1). Induction of the transport enzyme for each substrate, S i , is subject to positive feedback because the transport enzyme, E i , catalyses the accumulation of the inducer, X i which, in turn, stimulates the synthesis of even E i .…”

Section: Discussionmentioning

confidence: 99%

Quantitative effect and regulatory function of cyclic adenosine 5′-phosphate in Escherichia coli

2009

J Biosci

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Cyclic adenosine 5'-phosphate (cAMP) is a global regulator of gene expression in Escherichia coli. Despite decades of intensive study, the quantitative effect and regulatory function of cAMP remain the subjects of considerable debate. Here, we analyse the data in the literature to show that: (a) In carbon-limited cultures (including cultures limited by glucose), cAMP is at near-saturation levels with respect to expression of several catabolic promoters (including lac, ara and gal). It follows that cAMP receptor protein (CRP) cAMP-mediated regulation cannot account for the strong repression of these operons in the presence of glucose. (b) The cAMP levels in carbon-excess cultures are substantially lower than those observed in carbon-limited cultures under these conditions, the expression of catabolic promoters is very sensitive to variation of cAMP levels. (c)=CRPcAMP invariably activates the expression of catabolic promoters, but it appears to inhibit the expression of anabolic promoters. (d) These results suggest that the physiological function of cAMP is to maintain homeostatic energy levels. In carbon-limited cultures, growth is limited by the supply of energy; the cAMP levels therefore increase to enhance energy accumulation by activating the catabolic promoters and inhibiting the anabolic promoters. Conversely, in carbonexcess cultures, characterized by the availability of excess energy, the cAMP levels decrease in order to depress energy accumulation by inhibiting the catabolic promoters and activating the anabolic promoters.