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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 and fur mutant at C/N ratio 1.68 and 8.42.
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Figure 4: Comparison of the transcriptional mRNA levels between the wild type E. coli and fur mutant at C/N ratio 1.68 and 8.42.

Mentions: Figure 4 shows the effect of fur gene knockout on the RNA transcript levels, where it indicates that the transcript levels of PTS genes such as ptsH and ptsG, and the glycolysis genes such as pfkA and pykF as well as the oxidative pentose phosphate (PP) pathway genes such as zwf, gnd, and the ED pathway gene eda were all up-regulated as compared to wild type under N- rich condition (p < 0.01, p < 0.01; p < 0.01, p < 0.01; p < 0.01, p < 0.01, and p < 0.01, respectively) (Figure 4e). This is consistent with the fermentation data where the specific glucose consumption rate was higher for the mutant as compared to the wild type (Table 1). The activation of TCA cycle genes is consistent with the activation of the respiration where the transcript levels of cyoA, cydB, nuoA, ndh, atpA as well as sodA genes were all up-regulated (p < 0.01, p < 0.01, p < 0.01, p < 0.01, p < 0.01, and p < 0.01, respectively) (Figure 4f). Moreover, the transcript level of rpoN increased (p < 0.01), and the transcript levels of glnK, glnB, glnE were up-regulated (p < 0.05, p < 0.01, p < 0.01), and those of glnA, L, G, and gltB, D genes as well as nac gene were up-regulated (p < 0.01, p < 0.01, p < 0.01, and p < 0.01, p < 0.01, respectively), while gdhA gene transcript expression was decreased (p < 0.01)(Figure 4b). Those imply that GDH pathway was inactivated, while GS pathway was activated even under N-rich condition as also seen by enzyme activities (see Additional file 2).


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 and fur mutant 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 4: Comparison of the transcriptional mRNA levels between the wild type E. coli and fur mutant at C/N ratio 1.68 and 8.42.
Mentions: Figure 4 shows the effect of fur gene knockout on the RNA transcript levels, where it indicates that the transcript levels of PTS genes such as ptsH and ptsG, and the glycolysis genes such as pfkA and pykF as well as the oxidative pentose phosphate (PP) pathway genes such as zwf, gnd, and the ED pathway gene eda were all up-regulated as compared to wild type under N- rich condition (p < 0.01, p < 0.01; p < 0.01, p < 0.01; p < 0.01, p < 0.01, and p < 0.01, respectively) (Figure 4e). This is consistent with the fermentation data where the specific glucose consumption rate was higher for the mutant as compared to the wild type (Table 1). The activation of TCA cycle genes is consistent with the activation of the respiration where the transcript levels of cyoA, cydB, nuoA, ndh, atpA as well as sodA genes were all up-regulated (p < 0.01, p < 0.01, p < 0.01, p < 0.01, p < 0.01, and p < 0.01, respectively) (Figure 4f). Moreover, the transcript level of rpoN increased (p < 0.01), and the transcript levels of glnK, glnB, glnE were up-regulated (p < 0.05, p < 0.01, p < 0.01), and those of glnA, L, G, and gltB, D genes as well as nac gene were up-regulated (p < 0.01, p < 0.01, p < 0.01, and p < 0.01, p < 0.01, respectively), while gdhA gene transcript expression was decreased (p < 0.01)(Figure 4b). Those imply that GDH pathway was inactivated, while GS pathway was activated even under N-rich condition as also seen by enzyme activities (see Additional file 2).

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