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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

Transcription initiation sites and putative promoter regions. Transcription initiation sites and positions of putative σA, σB- and σ70/σS-dependent promoters upstream of ectA (A), ectB (B) and ectC (C). The -35 and -10 regions are underlined and the start codons ATG are framed. The transcription initiation sites as determined by RACE are typed bold, underlined twice and marked (+1). The DNA sequence upstream ectA which is deleted in pOSM16 (see text) is underlayed grey. The Sau3A restriction site used for the construction of pOSM2 (↱pOSM2) is marked. /◀◀: last nucleotide of the cDNA fragment from RACE experiment, which was terminated 89 bp upstream of the start codon of ectA (for E. coli) and 83 bp upstream of ectC (for M. halophilus) (see text).
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Figure 3: Transcription initiation sites and putative promoter regions. Transcription initiation sites and positions of putative σA, σB- and σ70/σS-dependent promoters upstream of ectA (A), ectB (B) and ectC (C). The -35 and -10 regions are underlined and the start codons ATG are framed. The transcription initiation sites as determined by RACE are typed bold, underlined twice and marked (+1). The DNA sequence upstream ectA which is deleted in pOSM16 (see text) is underlayed grey. The Sau3A restriction site used for the construction of pOSM2 (↱pOSM2) is marked. /◀◀: last nucleotide of the cDNA fragment from RACE experiment, which was terminated 89 bp upstream of the start codon of ectA (for E. coli) and 83 bp upstream of ectC (for M. halophilus) (see text).

Mentions: Putative transcription initiation sites for both, the donor (Marinococcus halophilus) and the genetically engineered acceptor (E. coli DH5α) were identified using the RACE technique. The identified sites are shown in Fig. 3. Subsequently potential promoter recognition sites were investigated by homology search using the Neural Network Promotor Prediction programme and assigned to the previously identified initiation sites. Primary eubacterial sigma factors are responsible for the transcription of most genes expressed in exponentially growing cells and essential for cell survival. They are known as σ70 in E. coli and σA in Bacillus subtilis and other Gram-positive bacteria and have identical consensus sequences. Non-essential stationary-phase or general stress response σ-factors of the Enterobacteriaceae are called σS and similar in function (but not in consensus sequence) to σB of B. subtilis and related organisms [25].


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

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

Transcription initiation sites and putative promoter regions. Transcription initiation sites and positions of putative σA, σB- and σ70/σS-dependent promoters upstream of ectA (A), ectB (B) and ectC (C). The -35 and -10 regions are underlined and the start codons ATG are framed. The transcription initiation sites as determined by RACE are typed bold, underlined twice and marked (+1). The DNA sequence upstream ectA which is deleted in pOSM16 (see text) is underlayed grey. The Sau3A restriction site used for the construction of pOSM2 (↱pOSM2) is marked. /◀◀: last nucleotide of the cDNA fragment from RACE experiment, which was terminated 89 bp upstream of the start codon of ectA (for E. coli) and 83 bp upstream of ectC (for M. halophilus) (see text).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Transcription initiation sites and putative promoter regions. Transcription initiation sites and positions of putative σA, σB- and σ70/σS-dependent promoters upstream of ectA (A), ectB (B) and ectC (C). The -35 and -10 regions are underlined and the start codons ATG are framed. The transcription initiation sites as determined by RACE are typed bold, underlined twice and marked (+1). The DNA sequence upstream ectA which is deleted in pOSM16 (see text) is underlayed grey. The Sau3A restriction site used for the construction of pOSM2 (↱pOSM2) is marked. /◀◀: last nucleotide of the cDNA fragment from RACE experiment, which was terminated 89 bp upstream of the start codon of ectA (for E. coli) and 83 bp upstream of ectC (for M. halophilus) (see text).
Mentions: Putative transcription initiation sites for both, the donor (Marinococcus halophilus) and the genetically engineered acceptor (E. coli DH5α) were identified using the RACE technique. The identified sites are shown in Fig. 3. Subsequently potential promoter recognition sites were investigated by homology search using the Neural Network Promotor Prediction programme and assigned to the previously identified initiation sites. Primary eubacterial sigma factors are responsible for the transcription of most genes expressed in exponentially growing cells and essential for cell survival. They are known as σ70 in E. coli and σA in Bacillus subtilis and other Gram-positive bacteria and have identical consensus sequences. Non-essential stationary-phase or general stress response σ-factors of the Enterobacteriaceae are called σS and similar in function (but not in consensus sequence) to σB of B. subtilis and related organisms [25].

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