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.