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Transcriptional regulation of main metabolic pathways of cyoA, cydB, fnr, and fur gene knockout Escherichia coli in C-limited and N-limited aerobic continuous cultures.

Kumar R, Shimizu K - Microb. Cell Fact. (2011)

Bottom Line: The present result is quite important in understanding the metabolic regulation for metabolic engineering.Moreover, the present result may be useful in improving the specific glucose consumption rate and activation of the TCA cycle by modulating the respiratory chain genes and the related global regulators.The result obtained under N-limited condition may be useful for the heterologous protein production under N-limitation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan.

ABSTRACT

Background: It is important to understand the cellular responses emanating from environmental perturbations to redesign the networks for practical applications. In particular, the carbon (C) metabolism, nitrogen (N) assimilation, and energy generation are by far important, where those are interconnected and integrated to maintain cellular integrity. In our previous study, we investigated the effect of C/N ratio on the metabolic regulation of gdhA, glnL, glt B,D mutants as well as wild type Escherichia coli (Kumar and Shimizu, MCF, 1-17, 9:8,2010), where it was shown that the transcript levels of cyoA and cydB which encode the terminal oxidases, fnr and fur which encode global regulators were significantly up-regulated under N-limited condition as compared to C-limited condition. In the present study, therefore, the effects of such single-gene knockout on the metabolic regulation were investigated to clarify the roles of those genes in the aerobic continuous culture at the dilution rate of 0.2 h(-1).

Results: The specific glucose consumption rates and the specific CO2 production rates of cyoA, cydB, fnr, and fur mutants were all increased as compared to the wild type under both C-limited and N-limited conditions. The former phenomenon was consistent with the up-regulations of the transcript levels of ptsG and ptsH, which are consistent with down-regulations of crp and mlc genes. Moreover, the increase in the specific glucose consumption rate was also caused by up-regulations of the transcript levels of pfkA, pykF and possibly zwf, where those are consistent with the down regulations of cra, crp and mlc genes. Moreover, the transcript levels of rpoN together with glnK, glnB, glnE were up-regulated, and thus the transcript levels of glnA,L,G, and gltB,D as well as nac were up-regulated, while gdhA was down-regulated. This implies the interconnection between cAMP-Crp and PII-Ntr systems. Moreover, cyoA, cydB, fnr and fur gene deletions up-regulated the transcript levels of respiration (nuoA, ndh, cyoA, cydB, and atpA) and the oxidative stress related genes such as soxR, S and sodA, where this was further enhanced under N-limitation. In the cases of cyoA and cydB mutants, arcA, fnr, fur, cydB (for cyoA mutant), and cyoA (for cydB mutant) genes were up-regulated, which may be due to incomplete oxidation of quinol. It was also shown that fur gene transcript level was up-regulated in accordance with the activation of respiratory chain genes. It was shown that the deletion of fur gene activated the enterobactin pathway.

Conclusion: The present result demonstrated how the fermentation characteristics could be explained by the transcript levels of metabolic pathway genes as well as global regulators in relation to the knockout of such single genes as cyoA, cydB, fnr, and fur, and clarified the complex gene network regulation in relation to glycolysis, TCA cycle, respiration, and N-regulated pathways. The present result is quite important in understanding the metabolic regulation for metabolic engineering. Moreover, the present result may be useful in improving the specific glucose consumption rate and activation of the TCA cycle by modulating the respiratory chain genes and the related global regulators. The result obtained under N-limited condition may be useful for the heterologous protein production under N-limitation.

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Comparison of the transcriptional mRNA levels between the wild type E. coli, cyoA and cydB mutant genes at C/N ratio 1.68 and 8.42.
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Figure 2: Comparison of the transcriptional mRNA levels between the wild type E. coli, cyoA and cydB mutant genes at C/N ratio 1.68 and 8.42.

Mentions: The relative transcript levels for cyoA and cydB mutants are given as compared to those of the wild type in Figure 2, where it indicates that the transcript levels of ptsG, ptsH, pfkA, pykF as well as zwf and eda were all up-regulated (p < 0.01, p < 0.01, p < 0.01, p < 0.01; p < 0.01, p < 0.01, respectively) in accordance with the increase in the specific glucose consumption rate for cyoA mutant as compared to the wild type (Figure 2e). This is consistent with the down-regulations of the transcript levels of cra (p < 0.05), crp (p < 0.01), and mlc (p < 0.01) genes (Figure 2a) (see Additional file 1). Figure 2e also shows that the transcript levels of lpdA, gltA, icdA, aceA, fumC, and sdhC were increased (p < 0.01, p < 0.01, p < 0.01, p < 0.05, p < 0.01, and p < 0.01, respectively) for cyoA mutant as compared to the wild type under N- rich condition. The increased TCA cycle activity caused higher specific CO2 production rate for the mutant as compared to the wild type, and affected respiration. Figure 2f indicates that cyoA gene knockout caused the transcript level of cydB gene to be up-regulated, and those of other respiratory chain genes such as nuoA, ndh, and atpA genes were also up-regulated (p < 0.01, p < 0.01, and p < 0.01, respectively). This is consistent with the up-regulations of soxR, S and sodA (p < 0.01, p < 0.01, and p < 0.01). Moreover, the increase in yfiD may be due to increase of arcA (p < 0.01). Note that aspC was down regulated (p < 0.01) and gadA was up-regulated (p < 0.01), which will be discussed in the discussion section. Figure 2b indicates that the transcript level of rpoN increased, which caused the transcript levels of glnB and glnK to be up-regulated (p < 0.01 and p < 0.01), and those of glnA, L, G and gltB, D genes were up-regulated (p < 0.01, p < 0.01, p < 0.01, and p < 0.01, p < 0.01, respectively). Moreover, the transcript level of nac gene increased (p < 0.01) and gdhA gene decreased (p < 0.01). Those imply that GDH pathway was inativated, while GS pathway was activated for cyoA mutant (as well as cydB mutant) even under N-rich condition (see Additional file 2). This phenomenon was also enhanced under N-limited condition (Figure 2d). Note that cyoA knockout caused rpoS and fur transcript levels to be up-regulated (p < 0.01 and p < 0.01) while fnr transcript level changed little (Figure 2a).


Transcriptional regulation of main metabolic pathways of cyoA, cydB, fnr, and fur gene knockout Escherichia coli in C-limited and N-limited aerobic continuous cultures.

Kumar R, Shimizu K - Microb. Cell Fact. (2011)

Comparison of the transcriptional mRNA levels between the wild type E. coli, cyoA and cydB mutant genes at C/N ratio 1.68 and 8.42.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037301&req=5

Figure 2: Comparison of the transcriptional mRNA levels between the wild type E. coli, cyoA and cydB mutant genes at C/N ratio 1.68 and 8.42.
Mentions: The relative transcript levels for cyoA and cydB mutants are given as compared to those of the wild type in Figure 2, where it indicates that the transcript levels of ptsG, ptsH, pfkA, pykF as well as zwf and eda were all up-regulated (p < 0.01, p < 0.01, p < 0.01, p < 0.01; p < 0.01, p < 0.01, respectively) in accordance with the increase in the specific glucose consumption rate for cyoA mutant as compared to the wild type (Figure 2e). This is consistent with the down-regulations of the transcript levels of cra (p < 0.05), crp (p < 0.01), and mlc (p < 0.01) genes (Figure 2a) (see Additional file 1). Figure 2e also shows that the transcript levels of lpdA, gltA, icdA, aceA, fumC, and sdhC were increased (p < 0.01, p < 0.01, p < 0.01, p < 0.05, p < 0.01, and p < 0.01, respectively) for cyoA mutant as compared to the wild type under N- rich condition. The increased TCA cycle activity caused higher specific CO2 production rate for the mutant as compared to the wild type, and affected respiration. Figure 2f indicates that cyoA gene knockout caused the transcript level of cydB gene to be up-regulated, and those of other respiratory chain genes such as nuoA, ndh, and atpA genes were also up-regulated (p < 0.01, p < 0.01, and p < 0.01, respectively). This is consistent with the up-regulations of soxR, S and sodA (p < 0.01, p < 0.01, and p < 0.01). Moreover, the increase in yfiD may be due to increase of arcA (p < 0.01). Note that aspC was down regulated (p < 0.01) and gadA was up-regulated (p < 0.01), which will be discussed in the discussion section. Figure 2b indicates that the transcript level of rpoN increased, which caused the transcript levels of glnB and glnK to be up-regulated (p < 0.01 and p < 0.01), and those of glnA, L, G and gltB, D genes were up-regulated (p < 0.01, p < 0.01, p < 0.01, and p < 0.01, p < 0.01, respectively). Moreover, the transcript level of nac gene increased (p < 0.01) and gdhA gene decreased (p < 0.01). Those imply that GDH pathway was inativated, while GS pathway was activated for cyoA mutant (as well as cydB mutant) even under N-rich condition (see Additional file 2). This phenomenon was also enhanced under N-limited condition (Figure 2d). Note that cyoA knockout caused rpoS and fur transcript levels to be up-regulated (p < 0.01 and p < 0.01) while fnr transcript level changed little (Figure 2a).

Bottom Line: The present result is quite important in understanding the metabolic regulation for metabolic engineering.Moreover, the present result may be useful in improving the specific glucose consumption rate and activation of the TCA cycle by modulating the respiratory chain genes and the related global regulators.The result obtained under N-limited condition may be useful for the heterologous protein production under N-limitation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan.

ABSTRACT

Background: It is important to understand the cellular responses emanating from environmental perturbations to redesign the networks for practical applications. In particular, the carbon (C) metabolism, nitrogen (N) assimilation, and energy generation are by far important, where those are interconnected and integrated to maintain cellular integrity. In our previous study, we investigated the effect of C/N ratio on the metabolic regulation of gdhA, glnL, glt B,D mutants as well as wild type Escherichia coli (Kumar and Shimizu, MCF, 1-17, 9:8,2010), where it was shown that the transcript levels of cyoA and cydB which encode the terminal oxidases, fnr and fur which encode global regulators were significantly up-regulated under N-limited condition as compared to C-limited condition. In the present study, therefore, the effects of such single-gene knockout on the metabolic regulation were investigated to clarify the roles of those genes in the aerobic continuous culture at the dilution rate of 0.2 h(-1).

Results: The specific glucose consumption rates and the specific CO2 production rates of cyoA, cydB, fnr, and fur mutants were all increased as compared to the wild type under both C-limited and N-limited conditions. The former phenomenon was consistent with the up-regulations of the transcript levels of ptsG and ptsH, which are consistent with down-regulations of crp and mlc genes. Moreover, the increase in the specific glucose consumption rate was also caused by up-regulations of the transcript levels of pfkA, pykF and possibly zwf, where those are consistent with the down regulations of cra, crp and mlc genes. Moreover, the transcript levels of rpoN together with glnK, glnB, glnE were up-regulated, and thus the transcript levels of glnA,L,G, and gltB,D as well as nac were up-regulated, while gdhA was down-regulated. This implies the interconnection between cAMP-Crp and PII-Ntr systems. Moreover, cyoA, cydB, fnr and fur gene deletions up-regulated the transcript levels of respiration (nuoA, ndh, cyoA, cydB, and atpA) and the oxidative stress related genes such as soxR, S and sodA, where this was further enhanced under N-limitation. In the cases of cyoA and cydB mutants, arcA, fnr, fur, cydB (for cyoA mutant), and cyoA (for cydB mutant) genes were up-regulated, which may be due to incomplete oxidation of quinol. It was also shown that fur gene transcript level was up-regulated in accordance with the activation of respiratory chain genes. It was shown that the deletion of fur gene activated the enterobactin pathway.

Conclusion: The present result demonstrated how the fermentation characteristics could be explained by the transcript levels of metabolic pathway genes as well as global regulators in relation to the knockout of such single genes as cyoA, cydB, fnr, and fur, and clarified the complex gene network regulation in relation to glycolysis, TCA cycle, respiration, and N-regulated pathways. The present result is quite important in understanding the metabolic regulation for metabolic engineering. Moreover, the present result may be useful in improving the specific glucose consumption rate and activation of the TCA cycle by modulating the respiratory chain genes and the related global regulators. The result obtained under N-limited condition may be useful for the heterologous protein production under N-limitation.

Show MeSH
Related in: MedlinePlus