Directed evolution of an artificial bifunctional enzyme, gamma-glutamyl kinase/gamma-glutamyl phosphate reductase, for improved osmotic tolerance of Escherichia coli transformants.
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.
Chen
Mingqing
M
College of Life Sciences, Wuhan University, 430072 Wuhan, China.
Cao
Junwei
J
Zheng
Congyi
C
Liu
Qing
Q
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000502:physiology*; D025461:Feedback, Physiological / Q000235:genetics; D050939:Gene Fusion; D050698:Glutamate-5-Semialdehyde Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009994:Osmolar Concentration; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011392:Proline / Q000096:biosynthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D011392:Proline; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase