Open AccessDirected evolution of an artificial bifunctional enzyme, γ‐glutamyl kinase/γ‐glutamyl phosphate reductase, for improved osmotic tolerance of Escherichia coli transformants
Author(s) -
Chen Mingqing,
Cao Junwei,
Zheng Congyi,
Liu Qing
Publication year - 2006
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2006.00397.x
Subject(s) - escherichia coli , phosphofructokinase 2 , biochemistry , complementation , bacillus subtilis , biology , mutant , enzyme , auxotrophy , proline , kinase , reductase , microbiology and biotechnology , amino acid , gene , bacteria , genetics
To produce the artificial bifunctional enzyme γ‐glutamyl kinase/γ‐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 γ‐glutamyl kinase leads to overproduction of proline by host cells. The mutated γ‐glutamyl kinase/γ‐glutamyl phosphate reductase enzyme was rendered about 100‐fold less sensitive to proline‐mediated feedback inhibition than the control.