title: 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.
authors: Bioprocess Development Center, Kyowa Hakko Bio Co., Ltd., Ibaraki, Japan. mikiro.hayashi@kyowa-kirin.co.jp
authors: Mikiro
authors: M
authors: Hayashi
authors: Kazuhiko
authors: K
authors: Tabata
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D053898:Biosynthetic Pathways; D027081:DNA Gyrase / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial / Q000235:genetics*; D005973:Glutamine / Q000378:metabolism*; D060847:Metabolic Engineering; D008826:Microbial Sensitivity Tests; D009154:Mutation; D010641:Phenotype; D013869:Thiolester Hydrolases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005973:Glutamine; D013869:Thiolester Hydrolases; C511807:YeiG protein, E coli; D027081:DNA Gyrase