Affiliation:
1. Bioprocess Development Center, Kyowa Hakko Bio Co., Ltd., Ibaraki, Japan
Abstract
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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
18 articles.
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