Abstract
Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor ...Cra is responsible for the growth burden of glycerol overproducing
E. coli
. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in
E. coli
.
The operation of the central metabolism is typically assumed to be deterministic, but dynamics and high connectivity of the metabolic network make it potentially prone to generating fluctuations. ...However, time-resolved measurements of metabolite levels in individual cells that are required to characterize such fluctuations remained a challenge, particularly in small bacterial cells. Here we use single-cell metabolite measurements based on Förster resonance energy transfer, combined with computer simulations, to explore the real-time dynamics of the metabolic network of Escherichia coli. We observe that steplike exposure of starved E. coli to glycolytic carbon sources elicits large periodic fluctuations in the intracellular concentration of pyruvate in individual cells. These fluctuations are consistent with predicted oscillatory dynamics of E. coli metabolic network, and they are primarily controlled by biochemical reactions around the pyruvate node. Our results further indicate that fluctuations in glycolysis propagate to other cellular processes, possibly leading to temporal heterogeneity of cellular states within a population.
Microbes must ensure robust amino acid metabolism in the face of external and internal perturbations. This robustness is thought to emerge from regulatory interactions in metabolic and genetic ...networks. Here, we explored the consequences of removing allosteric feedback inhibition in seven amino acid biosynthesis pathways in Escherichia coli (arginine, histidine, tryptophan, leucine, isoleucine, threonine, and proline). Proteome data revealed that enzyme levels decreased in five of the seven dysregulated pathways. Despite that, flux through the dysregulated pathways was not limited, indicating that enzyme levels are higher than absolutely needed in wild-type cells. We showed that such enzyme overabundance renders the arginine, histidine, and tryptophan pathways robust against perturbations of gene expression, using a metabolic model and CRISPR interference experiments. The results suggested a sensitive interaction between allosteric feedback inhibition and enzyme-level regulation that ensures robust yet efficient biosynthesis of histidine, arginine, and tryptophan in E. coli.
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•Amino acid biosynthesis enzymes do not normally operate at maximum capacity•Allosteric feedback inhibition ensures that enzymes are overabundant•Enzyme overabundance provides robustness against decreases in gene expression
Sander et al. explore the consequences of removing allosteric feedback inhibition in seven amino acid biosynthesis pathways in Escherichia coli. Their work suggests that allosteric feedback inhibition and transcriptional regulation of enzyme abundance work together to ensure robust yet efficient biosynthesis of histidine, arginine, and tryptophan.
Flow-injection mass spectrometry (FI-MS) enables metabolomics studies with a very high sample-throughput. However, FI-MS is prone to in-source modifications of analytes because samples are directly ...injected into the electrospray ionization source of a mass spectrometer without prior chromatographic separation. Here, we spiked authentic standards of 160 primary metabolites individually into an Escherichia coli metabolite extract and measured the thus derived 160 spike-in samples by FI-MS. Our results demonstrate that FI-MS can capture a wide range of chemically diverse analytes within 30 s measurement time. However, the data also revealed extensive in-source modifications. Across all 160 spike-in samples, we identified significant increases of 11,013 ion peaks in positive and negative mode combined. To explain these unknown m/z features, we connected them to the m/z feature of the (de-)protonated metabolite using information about mass differences and MS2 spectra. This resulted in networks that explained on average 49 % of all significant features. The networks showed that a single metabolite undergoes compound specific and often sequential in-source modifications like adductions, chemical reactions, and fragmentations. Our results show that FI-MS generates complex MS1 spectra, which leads to an overestimation of significant features, but neutral losses and MS2 spectra explain many of these features.
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•FI-MS enables measurements of chemically diverse metabolites.•Extensive in-source modifications during electrospray ionization are detected by FI-MS.•A network approach explains 49% of all recorded in-source modifications.
Enzymes maintain metabolism, and their concentration affects cellular fitness: high enzyme levels are costly, and low enzyme levels can limit metabolic flux. Here, we used CRISPR interference ...(CRISPRi) to study the consequences of decreasing E. coli enzymes below wild-type levels. A pooled CRISPRi screen with 7,177 strains demonstrates that metabolism buffers fitness defects for hours after the induction of CRISPRi. We characterized the metabolome and proteome responses in 30 CRISPRi strains and elucidated three gene-specific buffering mechanisms: ornithine buffered the knockdown of carbamoyl phosphate synthetase (CarAB) by increasing CarAB activity, S-adenosylmethionine buffered the knockdown of homocysteine transmethylase (MetE) by de-repressing expression of the methionine pathway, and 6-phosphogluconate buffered the knockdown of 6-phosphogluconate dehydrogenase (Gnd) by activating a bypass. In total, this work demonstrates that CRISPRi screens can reveal global sources of metabolic robustness and identify local regulatory mechanisms that buffer decreases of specific enzymes. A record of this paper’s transparent peer review process is included in the Supplemental Information.
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•An inducible CRISPRi system identifies rate-limiting enzymes•E. coli metabolism is robust against CRISPRi-knockdowns of enzymes•CRISPRi enforces specific metabolome and proteome responses•Regulatory metabolites buffer CRISPRi-knockdowns
Donati and Kuntz et al., study the consequences of CRISPR interference in Escherichia coli metabolism. Their work suggests that metabolism is robust against knockdowns of enzymes and that regulatory metabolite-protein interactions buffer such perturbations.
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