Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli.

Aiba, Hirofumi; Nakamura, Takanori; Mikami, Hiroshi; Mori, Hideki

doi:10.1002/j.1460-2075.1985.tb04084.x

Cited by 82 publications

(85 citation statements)

References 27 publications

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“…4), suggesting its limited application in a broad range of E. coli catalysts. Several cAMP-independent CRP mutants were discovered to release CCR to some extent (16,37). But cell growth is often stunted by crp mutations, and sugar coutilization is not efficient, which was also observed in this work (Fig.…”

Section: Discussionsupporting

confidence: 69%

“…This mutation was previously reported to alter allosteric regulation (16). Position 141 is part of a hinge important for the intramolecular transduction of the activation signal (35).…”

Section: Discussionmentioning

confidence: 79%

“…Theoretically, a cAMP-independent CRP variant should be able to activate the catabolic operons of secondary sugars even in the presence of glucose. However, these cAMP-independent CRP mutants often cannot activate the target operons at the same efficiency as wild-type CRP bound with cAMP (16). In addition, as an important global regulator, CRP directly regulates the transcriptional expression of more than 400 genes, and CRP mutants often have slow growth phenotypes potentially due to unpredictable expressional changes of other important genes (19).…”

Section: Significancementioning

confidence: 99%

“…CRP G141D was previously identified as a mutant with an altered allosteric mechanism, and position 141 is involved in the interfacial interactions between subunits and domains (16,27). Potentially due to the CRP G141D mutation in the evolved strain LP2, the transcription of xylose catabolic genes was up-regulated by more than 10-fold as measured by qRT-PCR (Fig.…”

Section: Characterization Of Physiological Effects Of Convergent Mutamentioning

confidence: 99%

“…Common strategies to engineer sugar coutilization in E. coli include the inactivation of PTS components, such as PtsG and PtsI (13)(14)(15), and mutagenesis of CRP (16,17).…”

mentioning

confidence: 99%

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Experimental evolution reveals an effective avenue to release catabolite repression via mutations in XylR

Sievert

Nieves

Panyon

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Microbial production of fuels and chemicals from lignocellulosic biomass provides promising biorenewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocellulosic biomass materials are glucose and xylose. Although industrial Escherichia coli strains efficiently use glucose, their ability to use xylose is often repressed in the presence of glucose. Here we independently evolved three E. coli strains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into the E. coli D-lactate producer TG114, 94% of a glucose–xylose mixture (50 g⋅L−1 each) was used in mineral salt media that led to a 50% increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in different E. coli hosts, including wild type, suggesting its potential wide application in industrial E. coli biocatalysts.

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Section: Discussionsupporting

confidence: 69%

“…This mutation was previously reported to alter allosteric regulation (16). Position 141 is part of a hinge important for the intramolecular transduction of the activation signal (35).…”

Section: Discussionmentioning

confidence: 79%

Section: Significancementioning

confidence: 99%

Section: Characterization Of Physiological Effects Of Convergent Mutamentioning

confidence: 99%

“…Common strategies to engineer sugar coutilization in E. coli include the inactivation of PTS components, such as PtsG and PtsI (13)(14)(15), and mutagenesis of CRP (16,17).…”

mentioning

confidence: 99%

See 3 more Smart Citations

Experimental evolution reveals an effective avenue to release catabolite repression via mutations in XylR

Sievert

Nieves

Panyon

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Experimental evolution reveals an effective avenue for d‐lactic acid production from glucose‐xylose mixtures via enhanced Glk activity and a cAMP‐independent CRP mutation

Qiao,

Fang,

et al. 2024

Biotech & Bioengineering

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Abstractd‐Lactic acid holds significant industrial importance due to its versatility and serves as a crucial component in the synthesis of environmentally friendly and biodegradable thermal‐resistant poly‐lactic acid. This polymer exhibits promising potential as a substitute for nonbiodegradable, petroleum‐based plastics. The production of d‐lactic acid from lignocellulosic biomass, a type of biorenewable and nonfood resources, can lower costs and improve product competitiveness. Glucose and xylose are the most abundant sugar monomers in lignocellulosic biomass materials. Despite Escherichia coli possessing native xylose catabolic pathways and transport, their ability to effectively utilize xylose is often hindered in the presence of glucose. Here, the E. coli strain Rec1.0, previously engineered to overcome carbon catabolite repression, was selected as the initial strain for reengineering to produce d‐lactic acid. An adaptive evolution approach was employed to achieve highly efficient fermentation of glucose‐xylose mixtures. The resulting strain, QJL010, could produce d‐lactic acid of 87.5 g/L with a carbon yield of 0.99 mol/mol. Notably, the consumption rates of glucose and xylose reached 0.75 and 0.82 g/gDCW/h, respectively. Further analysis revealed that increased Glk activity, resulting from glk mutations (A142V and R188H), along with their upregulated expression, contributed to an elevated glucose consumption rate. Additionally, a CRP G141D mutation, cAMP‐independent, stimulated the expression of the xylR, xylE, and galABC* genes, resulting in an accelerated xylose consumption rate. These findings provide valuable support for the utilization of E. coli platform strains in the production of value‐added chemicals from lignocellulosic biomass.

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Syn, anti, and finally both conformations of cyclic AMP are involved in the CRP‐dependent transcription initiation mechanism in E. coli lac operon

Tutar¹

2008

Cell Biochemistry & Function

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The cyclic AMP receptor protein (CRP) of Escherichia coli regulates the activity of more than 150 genes. Allosteric changes in CRP structure accompanied by cAMP binding, initiate transcription through protein binding to specific DNA sequences. Initially, researchers proposed a two-site cAMP-binding model for CRP-dependent transcription activation since biophysical methods showed two transitions during titration experiments. Three conformational states were considered; apo-CRP, CRP:(cAMP)(1) and CRP:(cAMP)(2), and CRP:(cAMP)(1) was proposed as the active form in this initial model. X-ray data indicated an anti conformation and in contrast NMR experiments suggested a syn conformation for bound cAMPs. For years this paradigm about ligand conformation has been ambiguous. When CRP was crystallized with four bound cAMP in the last decade, two cAMPs were assigned to syn and the other two to anti conformations. Again three conformational states were suggested; apo-CRP, CRP:(cAMP)(2), and CRP:(cAMP)(4). This new structure changed the view of CRP allosteric activation from a two-site model to a four-site model in the literature and the new model has been supported by biochemical and genetic data so far. According to the accepted model, binding of the first two cAMP molecules displays positive cooperativity, however, binding of the last two cAMP molecules shows negative cooperativity. This resolved the conflict between dynamic and static experimental observations. However, this new model cannot explain the initiation mechanism as previously proposed because functionally active CRP has only one cAMP equivalent. Gene regulation and transcription factors are involved in regulating both prokaryotic and eukaryotic metabolism. Although gene regulation and expression are much more complex in eukaryotes, CRP-mediated transcription initiation is a model of general interest to life sciences and medicine. Therefore, the aim of this review is to summarize recent works and developments on the cAMP-dependent CRP activation mechanism in E. coli.

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Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli.

Cited by 82 publications

References 27 publications

Experimental evolution reveals an effective avenue to release catabolite repression via mutations in XylR

Experimental evolution reveals an effective avenue to release catabolite repression via mutations in XylR

Experimental evolution reveals an effective avenue for d‐lactic acid production from glucose‐xylose mixtures via enhanced Glk activity and a cAMP‐independent CRP mutation

Syn, anti, and finally both conformations of cyclic AMP are involved in the CRP‐dependent transcription initiation mechanism in E. coli lac operon

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