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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

Construction of the plasmids pAKECT1. Plasmid pAKECT1 (10.1 kb) was constructed from pOSM12 and pRK1. Only donor plasmids, final construct and the relevant restriction sites are shown. Due to lack of suitable restriction sites, a complex construction scheme had to be applied (details in text). ectUp: region upstream of ectA with putative osmoregulated promoter sequences. lysC: deregulated aspartate kinase from Corynebacterium glutamicum MH20-22B.
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Figure 7: Construction of the plasmids pAKECT1. Plasmid pAKECT1 (10.1 kb) was constructed from pOSM12 and pRK1. Only donor plasmids, final construct and the relevant restriction sites are shown. Due to lack of suitable restriction sites, a complex construction scheme had to be applied (details in text). ectUp: region upstream of ectA with putative osmoregulated promoter sequences. lysC: deregulated aspartate kinase from Corynebacterium glutamicum MH20-22B.

Mentions: The plasmid pAKECT1 (Fig. 7) contains lysC under the control of a tac promoter and the gene cluster ectABC with the putative osmoregulated promoter sequence upstream of ectA (ectUp), just as in pOSM12. The possibility to separately induce the ectoine biosynthetic genes by osmotic stress and the aspartate kinase by IPTG enabled us to directly investigate the effect of the deregulated aspartate kinase on growth rates and ectoine production. As shown in Fig. 5, induction of aspartate kinase increased the growth rate at low salinity (1% and 2% NaCl) to the same level as the control. Without IPTG-induction of the feedback-insensitive aspartate kinase gene the ectoine levels in the cells were similar to those in DH5α pOSM12, but upon addition of IPTG, ectoine production increased approx. 3-fold in the range of 1–3% NaCl and reached a maximum of 0.4 mmol (g dry weight)-1, which remained relatively constant at salinities of 3% NaCl, to 5% NaCl (Fig. 8). The observed saturation level at 3% NaCl and higher perfectly correlated with the levels achieved by ectoine uptake from the growth medium (Fig. 4). This observation provides strong evidence that we succeeded in by-passing the regulatory mechanisms which caused the metabolic restriction for ectoine production in recombinant E. coli DH5α.


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

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

Construction of the plasmids pAKECT1. Plasmid pAKECT1 (10.1 kb) was constructed from pOSM12 and pRK1. Only donor plasmids, final construct and the relevant restriction sites are shown. Due to lack of suitable restriction sites, a complex construction scheme had to be applied (details in text). ectUp: region upstream of ectA with putative osmoregulated promoter sequences. lysC: deregulated aspartate kinase from Corynebacterium glutamicum MH20-22B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Construction of the plasmids pAKECT1. Plasmid pAKECT1 (10.1 kb) was constructed from pOSM12 and pRK1. Only donor plasmids, final construct and the relevant restriction sites are shown. Due to lack of suitable restriction sites, a complex construction scheme had to be applied (details in text). ectUp: region upstream of ectA with putative osmoregulated promoter sequences. lysC: deregulated aspartate kinase from Corynebacterium glutamicum MH20-22B.
Mentions: The plasmid pAKECT1 (Fig. 7) contains lysC under the control of a tac promoter and the gene cluster ectABC with the putative osmoregulated promoter sequence upstream of ectA (ectUp), just as in pOSM12. The possibility to separately induce the ectoine biosynthetic genes by osmotic stress and the aspartate kinase by IPTG enabled us to directly investigate the effect of the deregulated aspartate kinase on growth rates and ectoine production. As shown in Fig. 5, induction of aspartate kinase increased the growth rate at low salinity (1% and 2% NaCl) to the same level as the control. Without IPTG-induction of the feedback-insensitive aspartate kinase gene the ectoine levels in the cells were similar to those in DH5α pOSM12, but upon addition of IPTG, ectoine production increased approx. 3-fold in the range of 1–3% NaCl and reached a maximum of 0.4 mmol (g dry weight)-1, which remained relatively constant at salinities of 3% NaCl, to 5% NaCl (Fig. 8). The observed saturation level at 3% NaCl and higher perfectly correlated with the levels achieved by ectoine uptake from the growth medium (Fig. 4). This observation provides strong evidence that we succeeded in by-passing the regulatory mechanisms which caused the metabolic restriction for ectoine production in recombinant E. coli DH5α.

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