Abstract
To produce the artificial bifunctional enzyme gamma-glutamyl kinase/gamma-glutamyl phosphate reductase, a mutant library of the proBA fusion gene from Bacillus subtilis was created by error-prone PCR. Selecting by functional complementation of the proline auxotroph Escherichia coli JM83 and NaCl tolerance, we isolated a mutant of the proBA fusion gene that improved the osmotolerance of host cells of E. coli JM83. A single amino acid replacement (Asn177Asp) located in a conserved domain in gamma-glutamyl kinase leads to overproduction of proline by host cells. The mutated gamma-glutamyl kinase/gamma-glutamyl phosphate reductase enzyme was rendered about 100-fold less sensitive to proline-mediated feedback inhibition than the control.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Amino Acid Substitution
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Bacillus subtilis / enzymology
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Bacillus subtilis / genetics
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Directed Molecular Evolution*
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Escherichia coli / enzymology
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Escherichia coli / genetics*
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Escherichia coli / physiology*
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Feedback, Physiological / genetics
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Gene Fusion
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Glutamate-5-Semialdehyde Dehydrogenase / genetics*
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Glutamate-5-Semialdehyde Dehydrogenase / metabolism
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Molecular Sequence Data
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Mutagenesis
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Osmolar Concentration
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Phosphotransferases (Carboxyl Group Acceptor) / genetics*
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Phosphotransferases (Carboxyl Group Acceptor) / metabolism
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Polymerase Chain Reaction / methods
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Proline / biosynthesis
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Proline / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Sequence Alignment
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Sodium Chloride / metabolism
Substances
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Recombinant Fusion Proteins
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Sodium Chloride
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Proline
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Glutamate-5-Semialdehyde Dehydrogenase
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Phosphotransferases (Carboxyl Group Acceptor)
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glutamate 5-kinase