초록
<P>An <SMALL>l</SMALL>-glutamine-overproducing mutant of an <I>Escherichia coli</I> K-12-derived strain was selected from randomly mutagenized cells in the course of <SMALL>l</SMALL>-alanyl-<SMALL>l</SMALL>-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism for <SMALL>l</SMALL>-glutamine overproduction in this mutant. Three mutations were identified that are related to the <SMALL>l</SMALL>-glutamine overproduction phenotype, namely, an intergenic mutation in the 5′-flanking region of <I>yeiG</I> and two nonsynonymous mutations in <I>gyrA</I> (Gly821Ser and Asp830Asn). Expression of <I>yeiG</I>, which encodes a putative esterase, was enhanced by the intergenic mutation. The nonsynonymous mutations in <I>gyrA</I>, a gene that encodes the DNA gyrase α subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (<I>topA</I>) and topoisomerase IV (<I>parC</I> and <I>parE</I>), an increase in <SMALL>l</SMALL>-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase in <SMALL>l</SMALL>-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed an <SMALL>l</SMALL>-glutamine-hyperproducing strain by attenuating cellular <SMALL>l</SMALL>-glutamine degradation activity. Although the reconstituted mutant (with <I>yeiG</I> together with <I>gyrA</I>) produced 200 mM <SMALL>l</SMALL>-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mM <SMALL>l</SMALL>-glutamine.</P>