Metabolic Engineering for l -Glutamine Overproduction by Using DNA Gyrase Mutations in Escherichia coli

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