Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra)

Zhu, Liwen; Xia, Shitao; Wei, Lina; Li, Hongmei; Zhu, Yuan; Tang, Ya‐Jie

doi:10.1038/srep36526

Cited by 21 publications

(21 citation statements)

References 37 publications

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“…Other contaminants, also present in binding assays, could also have influenced previous experiments (Wei et al, ; Zhu et al, ). For instance, counter ions present in phosphorylated metabolite salt solutions introduced along with the ligand might affect electrostatic interactions involved in ligand–protein or protein–DNA contacts.…”

Section: Discussionmentioning

confidence: 99%

“…However, because of the significant differences between upper glycolysis in P. putida and E. coli the regulation of Cra in these organisms might be also different. More recently, mutants of Cra were reported to display affinities for FBP from nano to low micromolar range (Wei et al, ; Zhu et al, ). However, these authors could not find a significant effect of these mutations on either the regulation Cra’s DNA‐binding activity in vitro or on transcriptional control of its targets in vivo (Wei et al, ; Zhu et al, ), which casts doubts about the specificity and the functional consequences of the alleged change in Cra’s affinity for FBP.…”

Section: Introductionmentioning

confidence: 99%

“…More recently, mutants of Cra were reported to display affinities for FBP from nano to low micromolar range (Wei et al, ; Zhu et al, ). However, these authors could not find a significant effect of these mutations on either the regulation Cra’s DNA‐binding activity in vitro or on transcriptional control of its targets in vivo (Wei et al, ; Zhu et al, ), which casts doubts about the specificity and the functional consequences of the alleged change in Cra’s affinity for FBP. Therefore, although it has been widely assumed that FBP regulates Cra in E. coli (Ramseier et al, ; Ramseier et al, ; Saier and Ramseier, ; Kotte et al, ; Kochanowski et al, ; Chubukov et al, ; Lehning et al, ), a definite experimental and functionally relevant proof of the interaction between FBP and Cra is still missing.…”

Section: Introductionmentioning

confidence: 99%

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Assessment of the interaction between the flux‐signaling metabolite fructose‐1,6‐bisphosphate and the bacterial transcription factors CggR and Cra

Folly

Ortega

Hubmann

et al. 2018

Molecular Microbiology

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Bacteria regulate cell physiology in response to extra- and intracellular cues. Recent work showed that metabolic fluxes are reported by specific metabolites, whose concentrations correlate with flux through the respective metabolic pathway. An example of a flux-signaling metabolite is fructose-1,6-bisphosphate (FBP). In turn, FBP was proposed to allosterically regulate master regulators of carbon metabolism, Cra in Escherichia coli and CggR in Bacillus subtilis. However, a number of questions on the FBP-mediated regulation of these transcription factors is still open. Here, using thermal shift assays and microscale thermophoresis we demonstrate that FBP does not bind Cra, even at millimolar physiological concentration, and with electrophoretic mobility shift assays we also did not find FBP-mediated impairment of Cra's affinity for its operator site, while fructose-1-phosphate does. Furthermore, we show for the first time that FBP binds CggR within the millimolar physiological concentration range of the metabolite, and decreases DNA-binding activity of this transcription factor. Molecular docking experiments only identified a single FBP binding site CggR. Our results provide the long thought after clarity with regards to regulation of Cra activity in E. coli and reveals that E. coli and B. subtilis use distinct cellular mechanism to transduce glycolytic flux signals into transcriptional regulation.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Assessment of the interaction between the flux‐signaling metabolite fructose‐1,6‐bisphosphate and the bacterial transcription factors CggR and Cra

Folly

Ortega

Hubmann

et al. 2018

Molecular Microbiology

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“…To overcome the above limitations, researchers have tried to develop various metabolic engineering strategies to enhance SA production mostly in E. coli [4][5][6][7][8]. For example, yield was…”

Section: Introductionmentioning

confidence: 99%

Genome-Scale In Silico Analysis for Enhanced Production of Succinic Acid in Zymomonas mobilis

et al. 2018

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Abstract:Presented herein is a model-driven strategy for characterizing the production capability of expression host and subsequently identifying targets for strain improvement by resorting to network structural comparison with reference strain and in silico analysis of genome-scale metabolic model. The applicability of the strategy was demonstrated by exploring the capability of Zymomonas mobilis, as a succinic acid producer. Initially, the central metabolism of Z. mobilis was compared with reference producer, Mannheimia succiniciproducens, in order to identify gene deletion targets. It was followed by combinatorial gene deletion analysis. Remarkably, resultant in silico strains suggested that knocking out pdc, ldh, and pfl genes encoding pyruvate-consuming reactions as well as the cl gene leads to fifteen-fold increase in succinic acid molar yield. The current exploratory work could be a promising support to wet experiments by providing guidance for metabolic engineering strategies and lowering the number of trials and errors.

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“…Correspondingly, a higher Cra-FBP affinity ( K d = 130 ± 37 nM) was obtained, while the K d value of wild-type Cra with FBP was 1400 ± 150 nM. It suggested that the enhanced binding affinity of the Cra mutant for FBP may contribute to succinate biosynthesis [23]. However, research on how changes in effector binding affinity modulate DNA-binding and, therefore, the cell phenotype, is limited.…”

Section: Introductionmentioning

confidence: 99%

Succinate production positively correlates with the affinity of the global transcription factor Cra for its effector FBP in Escherichia coli

Wei

Zhu

Tang

2016

Biotechnol Biofuels

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BackgroundEffector binding is important for transcription factors, affecting both the pattern and function of transcriptional regulation to alter cell phenotype. Our previous work suggested that the affinity of the global transcription factor catabolite repressor/activator (Cra) for its effector fructose-1,6-bisphosphate (FBP) may contribute to succinate biosynthesis. To support this hypothesis, single-point and three-point mutations were proposed through the semi-rational design of Cra to improve its affinity for FBP.ResultsFor the first time, a positive correlation between succinate production and the affinity of Cra for FBP was revealed in Escherichia coli. Using the best-fit regression function, a cubic equation was used to examine and describe the relationship between succinate production and the affinity of Cra for FBP, demonstrating a significant positive correlation between the two factors (coefficient of determination R 2 = 0.894, P = 0.000 < 0.01). The optimal mutant strain was Tang1683, which provided the lowest mutation energy of −4.78 kcal/mol and the highest succinate concentration of 92.7 g/L, which was 34% higher than that obtained using an empty vector control. The parameters for the interaction between Cra and DNA showed that Cra bound to the promoter regions of pck and aceB to activate the corresponding genes. Normally, Cra-regulated operons under positive control are deactivated in the presence of FBP. Therefore, theoretically, the enhanced affinity of Cra for FBP will inhibit the activation of pck and aceB. However, the activation of genes involved in CO2 fixation and the glyoxylate pathway was further improved by the Cra mutant, ultimately contributing to succinate biosynthesis.ConclusionsEnhanced binding of Cra to FBP or active site mutations may eliminate the repressive effect caused by FBP, thus leading to increased activation of genes associated with succinate biosynthesis in the Cra mutant. This work demonstrates an important transcriptional regulation strategy in the metabolic engineering of succinate production and provides useful information for better understanding of the regulatory mechanisms of transcription factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0679-7) contains supplementary material, which is available to authorized users.

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Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra)

Cited by 21 publications

References 37 publications

Assessment of the interaction between the flux‐signaling metabolite fructose‐1,6‐bisphosphate and the bacterial transcription factors CggR and Cra

Assessment of the interaction between the flux‐signaling metabolite fructose‐1,6‐bisphosphate and the bacterial transcription factors CggR and Cra

Genome-Scale In Silico Analysis for Enhanced Production of Succinic Acid in Zymomonas mobilis

Succinate production positively correlates with the affinity of the global transcription factor Cra for its effector FBP in Escherichia coli

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