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Heterologous ectoine production in Escherichia coli: by-passing the metabolic bottle-neck.

Bestvater T, Louis P, Galinski EA - Saline Syst. (2008)

Bottom Line: Consequently, mRNA-fragments containing the single genes and combinations of the genes ectA and ectB or ectB and ectC, respectively, could be detected by Northern blot analysis.In addition, aspartate kinases were identified as the main limiting factor for ectoine production in recombinant E. coli DH5alpha.Co-expression of the ectoine biosynthesis genes and of the gene of the feedback-resistant aspartate kinase from Corynebacterium glutamicum MH20-22B (lysC) led to markedly increased production of ectoine in E. coli DH5alpha, resulting in cytoplasmic ectoine concentrations comparable to those reached via ectoine accumulation from the medium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemistry, Westfälische Wilhelms-Universität Münster, Münster, Germany. thorsten.bestvater.tb@bayermaterialscience.com

ABSTRACT
Transcription of the ectoine biosynthesis genes ectA, ectB and ectC from Marinococcus halophilus in recombinant Escherichia coli DH5alpha is probably initiated from three individual sigma70/sigmaA-dependent promoter sequences, upstream of each gene. Consequently, mRNA-fragments containing the single genes and combinations of the genes ectA and ectB or ectB and ectC, respectively, could be detected by Northern blot analysis. Under the control of its own regulatory promoter region (ectUp) a seemingly osmoregulated ectoine production was observed. In addition, aspartate kinases were identified as the main limiting factor for ectoine production in recombinant E. coli DH5alpha. Co-expression of the ectoine biosynthesis genes and of the gene of the feedback-resistant aspartate kinase from Corynebacterium glutamicum MH20-22B (lysC) led to markedly increased production of ectoine in E. coli DH5alpha, resulting in cytoplasmic ectoine concentrations comparable to those reached via ectoine accumulation from the medium.

No MeSH data available.


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Maximum growth rates. Maximum growth rates [h-1] of the recombinant ectoine producer E. coli DH5α pOSM12 (▲) and of the control strain E. coli DH5α pHSG575, with (●) and without (○) supplementation of 2 mM ectoine at salinities of between 1% and 5% NaCl in minimal medium MM63. The novel construct pAKECT1 (■) employing deregulated aspartate kinase from C. glutamicum (induced with 0.5 mM IPTG) displayed improved growth at salinities above 3% NaCl. Mean values and standard deviations are based on three independent experiments.
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Figure 5: Maximum growth rates. Maximum growth rates [h-1] of the recombinant ectoine producer E. coli DH5α pOSM12 (▲) and of the control strain E. coli DH5α pHSG575, with (●) and without (○) supplementation of 2 mM ectoine at salinities of between 1% and 5% NaCl in minimal medium MM63. The novel construct pAKECT1 (■) employing deregulated aspartate kinase from C. glutamicum (induced with 0.5 mM IPTG) displayed improved growth at salinities above 3% NaCl. Mean values and standard deviations are based on three independent experiments.

Mentions: Growth rates of DH5α pOSM2 were similar to those of the unsupplemented control strain DH5α pHSG575 without the ectoine biosynthetic genes (data not shown), whereas DH5α pOSM12 displayed slower growth than the control at salinities below 3% NaCl (Fig. 5). At salinities above 3% NaCl, however, a growth promoting effect was observed, which enabled the organism to tolerate up to 5% NaCl (Fig. 5). Although the intracellular concentration of ectoine increased linearly with salinity, it remained below ectoine levels achieved by the control strain DH5α pHSG575 through accumulation from the medium (Fig. 4). In the latter, ectoine accumulation increased up to a salinity of 3% to a final value of approx. 0.4 mmol (g dry weight)-1, which then remained constant at salinities between 3 and 5% NaCl, resulting in a strong promotion of cell growth (Fig. 5). Comparison with Halomonas elongata, an extremely halotolerant ectoine-producing bacterium, revealed that the intracellular ectoine levels were similar up to 3% NaCl but approximately 1.5-fold higher in H. elongata at 5% NaCl (data not shown). This indicates that E. coli DH5α, when grown in an ectoine-containing medium, is able to establish the required ectoine levels in a salinity range of 1–3% NaCl through uptake mechanism, but not at higher salinities. The heterologous ectoine production, on the other hand, appears to be restricted, leading to markedly lower ectoine levels in the cells (Fig. 4).


Heterologous ectoine production in Escherichia coli: by-passing the metabolic bottle-neck.

Bestvater T, Louis P, Galinski EA - Saline Syst. (2008)

Maximum growth rates. Maximum growth rates [h-1] of the recombinant ectoine producer E. coli DH5α pOSM12 (▲) and of the control strain E. coli DH5α pHSG575, with (●) and without (○) supplementation of 2 mM ectoine at salinities of between 1% and 5% NaCl in minimal medium MM63. The novel construct pAKECT1 (■) employing deregulated aspartate kinase from C. glutamicum (induced with 0.5 mM IPTG) displayed improved growth at salinities above 3% NaCl. Mean values and standard deviations are based on three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562377&req=5

Figure 5: Maximum growth rates. Maximum growth rates [h-1] of the recombinant ectoine producer E. coli DH5α pOSM12 (▲) and of the control strain E. coli DH5α pHSG575, with (●) and without (○) supplementation of 2 mM ectoine at salinities of between 1% and 5% NaCl in minimal medium MM63. The novel construct pAKECT1 (■) employing deregulated aspartate kinase from C. glutamicum (induced with 0.5 mM IPTG) displayed improved growth at salinities above 3% NaCl. Mean values and standard deviations are based on three independent experiments.
Mentions: Growth rates of DH5α pOSM2 were similar to those of the unsupplemented control strain DH5α pHSG575 without the ectoine biosynthetic genes (data not shown), whereas DH5α pOSM12 displayed slower growth than the control at salinities below 3% NaCl (Fig. 5). At salinities above 3% NaCl, however, a growth promoting effect was observed, which enabled the organism to tolerate up to 5% NaCl (Fig. 5). Although the intracellular concentration of ectoine increased linearly with salinity, it remained below ectoine levels achieved by the control strain DH5α pHSG575 through accumulation from the medium (Fig. 4). In the latter, ectoine accumulation increased up to a salinity of 3% to a final value of approx. 0.4 mmol (g dry weight)-1, which then remained constant at salinities between 3 and 5% NaCl, resulting in a strong promotion of cell growth (Fig. 5). Comparison with Halomonas elongata, an extremely halotolerant ectoine-producing bacterium, revealed that the intracellular ectoine levels were similar up to 3% NaCl but approximately 1.5-fold higher in H. elongata at 5% NaCl (data not shown). This indicates that E. coli DH5α, when grown in an ectoine-containing medium, is able to establish the required ectoine levels in a salinity range of 1–3% NaCl through uptake mechanism, but not at higher salinities. The heterologous ectoine production, on the other hand, appears to be restricted, leading to markedly lower ectoine levels in the cells (Fig. 4).

Bottom Line: Consequently, mRNA-fragments containing the single genes and combinations of the genes ectA and ectB or ectB and ectC, respectively, could be detected by Northern blot analysis.In addition, aspartate kinases were identified as the main limiting factor for ectoine production in recombinant E. coli DH5alpha.Co-expression of the ectoine biosynthesis genes and of the gene of the feedback-resistant aspartate kinase from Corynebacterium glutamicum MH20-22B (lysC) led to markedly increased production of ectoine in E. coli DH5alpha, resulting in cytoplasmic ectoine concentrations comparable to those reached via ectoine accumulation from the medium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemistry, Westfälische Wilhelms-Universität Münster, Münster, Germany. thorsten.bestvater.tb@bayermaterialscience.com

ABSTRACT
Transcription of the ectoine biosynthesis genes ectA, ectB and ectC from Marinococcus halophilus in recombinant Escherichia coli DH5alpha is probably initiated from three individual sigma70/sigmaA-dependent promoter sequences, upstream of each gene. Consequently, mRNA-fragments containing the single genes and combinations of the genes ectA and ectB or ectB and ectC, respectively, could be detected by Northern blot analysis. Under the control of its own regulatory promoter region (ectUp) a seemingly osmoregulated ectoine production was observed. In addition, aspartate kinases were identified as the main limiting factor for ectoine production in recombinant E. coli DH5alpha. Co-expression of the ectoine biosynthesis genes and of the gene of the feedback-resistant aspartate kinase from Corynebacterium glutamicum MH20-22B (lysC) led to markedly increased production of ectoine in E. coli DH5alpha, resulting in cytoplasmic ectoine concentrations comparable to those reached via ectoine accumulation from the medium.

No MeSH data available.


Related in: MedlinePlus