scholarly journals Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure (R)-(−)-Hydroxycarboxylic Acids

2003 ◽  
Vol 69 (6) ◽  
pp. 3421-3426 ◽  
Author(s):  
Sang Yup Lee ◽  
Young Lee
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Ralstonia Eutropha ◽  
Enantiomerically Pure ◽  
Pha Depolymerase ◽  
E Coli ◽  
Theoretical Yield ◽  
Hydroxycarboxylic Acids ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid

ABSTRACT A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.

scholarly journals Phosphoethanolamine substitution in the lipid A of Escherichia coli O157 : H7 and its association with PmrC

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 657-666 ◽  
Author(s):  
Sang-Hyun Kim ◽  
Wenyi Jia ◽  
Valeria R. Parreira ◽  
Russell E. Bishop ◽  
Carlton L. Gyles
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Lipid A ◽  
High Copy Number ◽  
Peptide Antibiotics ◽  
Significant Similarity ◽  
E Coli ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid ◽  
K 12

This study shows that lipid A of Escherichia coli O157 : H7 differs from that of E. coli K-12 in that it has a phosphoform at the C-1 position, which is distinctively modified by a phosphoethanolamine (PEtN) moiety, in addition to the diphosphoryl form. The pmrC gene responsible for the addition of PEtN to the lipid A of E. coli O157 : H7 was inactivated and the changes in lipid A profiles were assessed. The pmrC null mutant still produced PEtN-modified lipid A species, albeit in a reduced amount, indicating that PmrC was not the only enzyme that could be used to add PEtN to lipid A. Natural PEtN substitution was shown to be present in the lipid A of other serotypes of enterohaemorrhagic E. coli and absent from the lipid A of E. coli K-12. However, the cloned pmrC O157 gene in a high-copy-number plasmid generated a large amount of PEtN-substituted lipid A species in E. coli K-12. The occurrence of PEtN-substituted lipid A species was associated with a slight increase in the MICs of cationic peptide antibiotics, suggesting that the lipid A modification with PEtN would be beneficial for survival of E. coli O157 : H7 in certain environmental niches. However, PEtN substitution in the lipid A profiles was not detected when putative inner-membrane proteins (YhbX/YbiP/YijP/Ecf3) that show significant similarity with PmrC in amino acid sequence were expressed from high-copy-number plasmids in E. coli K-12. This suggests that these potential homologues are not responsible for the addition of PEtN to lipid A in the pmrC mutant of E. coli O157 : H7. When cells were treated with EDTA, the amount of palmitoylated lipid A from the cells carrying a high-copy-number plasmid clone of pmrC O157 that resulted in significant increase of PEtN substitution was unchanged compared with cells without PEtN substitution, suggesting that the PEtN moiety substituted in lipid A does not compensate for the loss of divalent cations required for bridging neighbouring lipid A molecules.

scholarly journals Escherichia coli abg Genes Enable Uptake and Cleavage of the Folate Catabolite p-Aminobenzoyl-Glutamate

2007 ◽  
Vol 189 (9) ◽  
pp. 3329-3334 ◽  
Author(s):  
Eric L. Carter ◽  
Lindsey Jager ◽  
Lars Gardner ◽  
Christel C. Hall ◽  
Stacey Willis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Physiological Role ◽  
Structural Protein ◽  
Rapid Loss ◽  
Endogenous Expression ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid ◽  
Low Levels

ABSTRACT Escherichia coli AbgT was first identified as a structural protein enabling the growth of p-aminobenzoate auxotrophs on exogenous p-aminobenzoyl-glutamate (M. J. Hussein, J. M. Green, and B. P. Nichols, J. Bacteriol. 180:6260-6268, 1998). The abg region includes abgA, abgB, abgT, and ogt; these genes may be regulated by AbgR, a divergently transcribed LysR-type protein. Wild-type cells transformed with a high-copy-number plasmid encoding abgT demonstrate saturable uptake of p-aminobenzoyl-glutamate (KT = 123 μM); control cells expressing vector demonstrate negligible uptake. The addition of metabolic poisons inhibited uptake of p-aminobenzoyl-glutamate, consistent with this process requiring energy. p-Aminobenzoyl-glutamate taken in by cells expressing large amounts of AbgT alone is not rapidly metabolized to a form that is trapped in the cell, as the addition of nonradioactive p-aminobenzoyl-glutamate to these cells results in a rapid loss of intracellular label. The addition of nonradioactive p-aminobenzoate has no effect. The abgA, abgB, and abgAB genes were cloned into the medium-copy-number plasmid pACYC184; p-aminobenzoate auxotrophs transformed with the clone encoding abgAB demonstrated enhanced ability to grow on low levels of p-aminobenzoyl-glutamate. When transformed with complementary plasmids encoding high-copy levels of abgT and medium-copy levels of abgAB, p-aminobenzoate auxotrophs grew on 50 nM p-aminobenzoyl-glutamate. Our data are consistent with a model of p-aminobenzoyl-glutamate utilization in which AbgT catalyzes transport of p-aminobenzoyl-glutamate, followed by cleavage to p-aminobenzoate by a protein composed of subunits encoded by abgA and abgB. While endogenous expression of these genes is very low under the conditions in which we performed our experiments, these genes may be induced by AbgR bound to an unknown molecule. The true physiological role of this region may be related to some molecule similar to p-aminobenzoyl-glutamate, such as a dipeptide.

Complete sequence of low-copy-number plasmid MccC7-H22 of probiotic Escherichia coli H22 and the prevalence of mcc genes among human E. coli

Plasmid ◽  
2008 ◽  
Vol 59 (1) ◽  
pp. 1-10 ◽  
Author(s):  
David Šmajs ◽  
Michal Strouhal ◽  
Petra Matějková ◽  
Darina Čejková ◽  
Luciana Cursino ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Complete Sequence ◽  
E Coli ◽  
Copy Number Plasmid ◽  
Low Copy Number

scholarly journals The phasmid as a tool for plasmid genetics: II. Isolation of point mutations that affect replication of a ColE1-related plasmid

1982 ◽  
Vol 40 (3) ◽  
pp. 233-247 ◽  
Author(s):  
Gianni Cesareni ◽  
Luisa Castagnoli ◽  
Sydney Brenner
Keyword(s):  
Copy Number ◽  
Point Mutations ◽  
Plasmid Replication ◽  
Single Base ◽  
Lambdoid Phage ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid ◽  
Single Base Pair ◽  
Related Plasmid ◽  
Single Base Pair Change

SUMMARYThe insertion of a high-copy-number plasmid into a lambdoid phage chromosome which lacks a functional repressor gene confers on the hybrid ‘phasmid’ the capacity to grow on an immune lysogen. This was found to be due to titration of repressor because of plasmid replication. We have exploited this property in order to isolate mutants that affect plasmid replication. These mutants have been mapped in a region that was previously characterized as necessary for plasmid replication and incompatibility properties. Some of the mutations could revert at frequencies characteristic of single-base-pair change mutations.

scholarly journals Nucleotide sequence of a high copy number plasmid fromHalobacteriumstrain GRB

1990 ◽  
Vol 18 (11) ◽  
pp. 3408-3408 ◽  
Author(s):  
Neil R. Hackett ◽  
Mark P. Krebs ◽  
Shiladitya DasSarma ◽  
Werner Goebel ◽  
Uttam L. RajBhandary ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Copy Number ◽  
High Copy Number ◽  
Copy Number Plasmid ◽  
High Copy Number Plasmid
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