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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.


Related in: MedlinePlus

Intracellular ectoine content (heterologous production vs. uptake). Intracellular ectoine concentrations of the recombinant ectoine producers E. coli DH5α pOSM12 (black bars) and pOSM2 (grey bars), the latter supplemented with IPTG for induction of the lac promoter upstream of ectA, and of the control strain E. coli DH5α pHSG575, supplemented with 2 mM ectoine in the growth medium, (white bars) at salinities between 1% and 5% NaCl in minimal medium MM63. Mean values and standard deviations are based on three independent experiments.
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Figure 4: Intracellular ectoine content (heterologous production vs. uptake). Intracellular ectoine concentrations of the recombinant ectoine producers E. coli DH5α pOSM12 (black bars) and pOSM2 (grey bars), the latter supplemented with IPTG for induction of the lac promoter upstream of ectA, and of the control strain E. coli DH5α pHSG575, supplemented with 2 mM ectoine in the growth medium, (white bars) at salinities between 1% and 5% NaCl in minimal medium MM63. Mean values and standard deviations are based on three independent experiments.

Mentions: Plasmid pOSM12 carries a 3.5 kb DNA fragment from M. halophilus containing the ectoine biosynthetic genes ectABC and a putative promoter region (ectUp) 720 bp upstream of ectA, whereas on pOSM2 this upstream region is deleted 100 bp short of ectA and substituted by a lac promoter (Fig. 1B). Expression of the ectoine biosynthetic genes ectABC in E. coli DH5α using both pOSM12 and pOSM2, the latter supplemented with IPTG, led to an accumulation of ectoine in the cells (Fig. 4). The amount of intracellular ectoine increased with salinity of the growth medium in both cases but appeared to be slightly lower in the strain DH5α pOSM2. The most obvious difference was that the E. coli construct devoid of the promoter region (pOSM2) was unable to grow at 5% NaCl (Fig. 4). The levels of ectoine accumulation were, however, always lower than those of the control strain (DH5α pHSG575) in the presence of externally supplied ectoine (Fig. 4).


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

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

Intracellular ectoine content (heterologous production vs. uptake). Intracellular ectoine concentrations of the recombinant ectoine producers E. coli DH5α pOSM12 (black bars) and pOSM2 (grey bars), the latter supplemented with IPTG for induction of the lac promoter upstream of ectA, and of the control strain E. coli DH5α pHSG575, supplemented with 2 mM ectoine in the growth medium, (white bars) at salinities between 1% and 5% NaCl in minimal medium MM63. 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 4: Intracellular ectoine content (heterologous production vs. uptake). Intracellular ectoine concentrations of the recombinant ectoine producers E. coli DH5α pOSM12 (black bars) and pOSM2 (grey bars), the latter supplemented with IPTG for induction of the lac promoter upstream of ectA, and of the control strain E. coli DH5α pHSG575, supplemented with 2 mM ectoine in the growth medium, (white bars) at salinities between 1% and 5% NaCl in minimal medium MM63. Mean values and standard deviations are based on three independent experiments.
Mentions: Plasmid pOSM12 carries a 3.5 kb DNA fragment from M. halophilus containing the ectoine biosynthetic genes ectABC and a putative promoter region (ectUp) 720 bp upstream of ectA, whereas on pOSM2 this upstream region is deleted 100 bp short of ectA and substituted by a lac promoter (Fig. 1B). Expression of the ectoine biosynthetic genes ectABC in E. coli DH5α using both pOSM12 and pOSM2, the latter supplemented with IPTG, led to an accumulation of ectoine in the cells (Fig. 4). The amount of intracellular ectoine increased with salinity of the growth medium in both cases but appeared to be slightly lower in the strain DH5α pOSM2. The most obvious difference was that the E. coli construct devoid of the promoter region (pOSM2) was unable to grow at 5% NaCl (Fig. 4). The levels of ectoine accumulation were, however, always lower than those of the control strain (DH5α pHSG575) in the presence of externally supplied ectoine (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