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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

Ectoine biosynthesis and ectABC gene cluster from Marinococcus halophilus. A: The biosynthetic pathway for ectoine [56,57] and its dependence on feed-back regulation and/or transcriptional repression of the aspartate kinases in the biosynthetic pathway of the amino acids L-lysine, L-threonine and L-methionine during heterologous expression in E. coli. B: Map of the ectoine biosynthetic genes from M. halophilus as integrated in the plasmids pOSM12 and pOSM2 (only some restriction sites are shown). In case of pOSM2 the natural promoter region upstream of ectA is truncated and replaced by a lac promoter. 1, L-aspartate-kinase I-III; 2, L-aspartate-β-semialdehyde dehydrogenase; 3, L-2,4-diaminobutyric acid transaminase (ectB); 4, L-2,4-diaminobutyric acid Nγ-acetyltransferase (ectA); 5, L-ectoine synthase (ectC).
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Figure 1: Ectoine biosynthesis and ectABC gene cluster from Marinococcus halophilus. A: The biosynthetic pathway for ectoine [56,57] and its dependence on feed-back regulation and/or transcriptional repression of the aspartate kinases in the biosynthetic pathway of the amino acids L-lysine, L-threonine and L-methionine during heterologous expression in E. coli. B: Map of the ectoine biosynthetic genes from M. halophilus as integrated in the plasmids pOSM12 and pOSM2 (only some restriction sites are shown). In case of pOSM2 the natural promoter region upstream of ectA is truncated and replaced by a lac promoter. 1, L-aspartate-kinase I-III; 2, L-aspartate-β-semialdehyde dehydrogenase; 3, L-2,4-diaminobutyric acid transaminase (ectB); 4, L-2,4-diaminobutyric acid Nγ-acetyltransferase (ectA); 5, L-ectoine synthase (ectC).

Mentions: The non-halophilic Escherichia coli has been shown to accumulate ectoine from the surrounding medium, and as a consequence its tolerance to elevated salinities is increased [23]. Also, recombinant E. coli XL1-Blue is able to express the ectoine genes ectABC from the Gram-positive moderately halophilic Marinococcus halophilus and exploit the enzymes of the biosynthetic pathway for osmoregulated ectoine production [7]. The organization of the ectoine gene cluster and its relation to the ectoine biosynthetic pathway is shown in Fig. 1A and 1B. A search for consensus sequences for σ70/σA-dependent promoters revealed two potential promoter sites upstream of ectB, but none at the beginning of the gene cluster [7]. Using deletion derivatives, however, the authors were able to conclude that regulating sequences must extend up to or beyond 150 bp upstream of ectA.


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

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

Ectoine biosynthesis and ectABC gene cluster from Marinococcus halophilus. A: The biosynthetic pathway for ectoine [56,57] and its dependence on feed-back regulation and/or transcriptional repression of the aspartate kinases in the biosynthetic pathway of the amino acids L-lysine, L-threonine and L-methionine during heterologous expression in E. coli. B: Map of the ectoine biosynthetic genes from M. halophilus as integrated in the plasmids pOSM12 and pOSM2 (only some restriction sites are shown). In case of pOSM2 the natural promoter region upstream of ectA is truncated and replaced by a lac promoter. 1, L-aspartate-kinase I-III; 2, L-aspartate-β-semialdehyde dehydrogenase; 3, L-2,4-diaminobutyric acid transaminase (ectB); 4, L-2,4-diaminobutyric acid Nγ-acetyltransferase (ectA); 5, L-ectoine synthase (ectC).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Ectoine biosynthesis and ectABC gene cluster from Marinococcus halophilus. A: The biosynthetic pathway for ectoine [56,57] and its dependence on feed-back regulation and/or transcriptional repression of the aspartate kinases in the biosynthetic pathway of the amino acids L-lysine, L-threonine and L-methionine during heterologous expression in E. coli. B: Map of the ectoine biosynthetic genes from M. halophilus as integrated in the plasmids pOSM12 and pOSM2 (only some restriction sites are shown). In case of pOSM2 the natural promoter region upstream of ectA is truncated and replaced by a lac promoter. 1, L-aspartate-kinase I-III; 2, L-aspartate-β-semialdehyde dehydrogenase; 3, L-2,4-diaminobutyric acid transaminase (ectB); 4, L-2,4-diaminobutyric acid Nγ-acetyltransferase (ectA); 5, L-ectoine synthase (ectC).
Mentions: The non-halophilic Escherichia coli has been shown to accumulate ectoine from the surrounding medium, and as a consequence its tolerance to elevated salinities is increased [23]. Also, recombinant E. coli XL1-Blue is able to express the ectoine genes ectABC from the Gram-positive moderately halophilic Marinococcus halophilus and exploit the enzymes of the biosynthetic pathway for osmoregulated ectoine production [7]. The organization of the ectoine gene cluster and its relation to the ectoine biosynthetic pathway is shown in Fig. 1A and 1B. A search for consensus sequences for σ70/σA-dependent promoters revealed two potential promoter sites upstream of ectB, but none at the beginning of the gene cluster [7]. Using deletion derivatives, however, the authors were able to conclude that regulating sequences must extend up to or beyond 150 bp upstream of ectA.

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