期刊
FEMS MICROBIOLOGY LETTERS
卷 263, 期 1, 页码 41-47出版社
OXFORD UNIV PRESS
DOI: 10.1111/j.1574-6968.2006.00397.x
关键词
error-prone PCR; proBA fusion gene; proline biosynthesis; osmotic stress
类别
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.
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