A Simple Method for Multiple Modification of the<i>Escherichia coli</i>K-12 Chromosome

Mizoguchi, Hiroshi; Tanaka-Masuda, Kimie; Mori, Hideo

doi:10.1271/bbb.70274

Cited by 22 publications

(16 citation statements)

References 15 publications

(11 reference statements)

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“…Markerless gene deletion using the double-crossover method has been achieved in various bacteria (19,28,31,35,38). Generally, a low frequency of the second crossover event is an obstacle to successful gene deletion, and strategies for efficiently selecting second-crossover recombinants are required.…”

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confidence: 99%

Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation

Hirayama

Sakanaka

Fukuma

et al. 2012

Appl Environ Microbiol

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ABSTRACTFunctional analysis ofBifidobacteriumgenes is essential for understanding host-Bifidobacteriuminteractions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover inBifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-ΔrepA, which lacks the plasmid replication generepA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harborsrepA, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected inB. longum105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using theagagene, which encodes α-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion ofaga. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-basedaga-complemented strain. These functional analyses revealed thatagais the only gene encoding a functional α-galactosidase enzyme inB. longum105-A.

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confidence: 99%

Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation

Hirayama

Sakanaka

Fukuma

et al. 2012

Appl Environ Microbiol

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“…Strain JKYP9 was treated with N-methyl-N=-nitro-N-nitrosoguanidine (7), and strain M4 was isolated by selecting the L-glutamine producer from L-glutamine analog-resistant mutants. Markerless gene deletion and introduction of point mutations were performed using the modified phage lambda Red recombinase system (8,10). The primers used in this study are listed in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

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

Hayashi

Tabata

2013

Appl Environ Microbiol

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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 Lglutamine 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 ␣ 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. L-Glutamine, which is a semiessential amino acid in humans, is involved in biosynthesis of amino acids, nucleotides, and cofactors as an amido donor for glutamine amidotransferase in archaeal, bacterial, and eukaryotic cells. In mammalian cell cultures, L-glutamine is commonly added to the media as a nitrogen source (1). For medical applications, it is useful for treating injuries and gastric ulcers (2). We have already reported a novel method using L-amino acid ␣-ligase for fermentative production of L-alanyl-Lglutamine (Ala-Gln), an L-glutamine-containing dipeptide (3). In the course of development of the Ala-Gln-producing Escherichia coli strain, we found that enhancement of the metabolic flux with respect to substrate amino acids (i.e., L-alanine and L-glutamine) was essential for Ala-Gln production. In order to increase the Lglutamine supply in E. coli cells, two genes responsible for the regulation of L-glutamine biosynthesis were deleted from chromosome. One such gene, glnE, encodes glutamine synthetase (GS) adenylyltransferase, and the other, glnB, encodes PII protein. Deletion of glnE resulted in enhanced GS activity by preventing inactive GS-AMP formation. Furthermore, transcription of the glnA gene, encoding GS, was increased via the NtrB/NtrC regulatory system as a result of deleting glnB. We sought an alternative way of enhancing the metabolic flux for L-glutamine and have isolated an L-glutamine-overproducing mutant by random mutation and selection from the parental strain, JKYP9 (3). The genetic background t...

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“…13) MGF-01 ÁrspA was constructed by replacing ydfE, insD, intQ, rspB, and rspA by the sacB cat cassette. 8) The left arm region for homologous recombination was amplified using a 5 0 primer, 1646526U rsp Lch (5 0 -GTCAGCATGGATACTATCGATCTTG-3 0 ), and a 3 0 primer, 1648055C rsp Lcm (5 0 -GTGCTACGCCTGAATAA-GTGCGGCCGCTTGCTGGCATATAAGAATGAAACCG-3 0 ), and the right arm region was amplified using a 5 0 primer, 1654860C rsp Rch (5 0 -GTGGACTGTCAGCTAATCATTGTTG-3 0 ), and a 3 0 primer, 1653311U rsp Rsa (5 0 -CGCAAAAGAAAATGCCGATTTGCGGCC-GCAGAATACCTTTTGTCAGCTGACACT-3 0 ). These two fragments and the sacB cat cassette were used as templates for the next PCR, and a 5 0 primer, 1647046U rsp Lar (5 0 -TGATTACATACCCGCGTTAG-TAGCT-3 0 ), and a 3 0 primer, 1654355C rsp Rar (5 0 -TATCATACA-GATCAGAGAGTTCAAC-3 0 ), were used for amplification (nested PCR).…”

Section: Methodsmentioning

confidence: 99%

“…[3][4][5][6] We have constructed a reduced genome strain by a unique strategy. 7,8) Regions with more than 10 continuous genes which were deduced to be unnecessary regions for healthy growth in M9 minimal medium were chosen as candidates for deletion. The influence of each deletion on growth in M9 minimal medium was tested, and regions whose deletion did not affect growth were tested in combination.…”

mentioning

confidence: 99%

YdfH Identified as a Repressor ofrspAby the Use of Reduced GenomeEscherichia coliMGF-01

Sakihama¹,

Mizoguchi²,

Oshima³

et al. 2012

Bioscience, Biotechnology, and Biochemistry

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We have reported the construction of 1 Mb reduced genome Escherichia coli MGF-01 by a 28-step operation. This time, transcriptome analysis of MGF-01 was performed. Although the transcriptome profiles of the exponential phase in parental strain W3110red were well-conserved in MGF-01, the rspAB operon was highly expressed. A LacZ reporter assay of a series of stepwise deletion strains prepared in the course of MGF-01 construction indicated that rspA was highly expressed after the 5th step. Further analysis indicated that Á29, one of the deleted regions at the 5th step, relates to an increase in rspA expression, and that transcriptional regulator ydfH, in the Á29 region, is responsible for the expression of rspA, gel shift assay indicated that YdfH bound directly to the upstream region of rspA. Based on these results, it was concluded that YdfH is a transcriptional repressor of the rspAB operon.

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A Simple Method for Multiple Modification of theEscherichia coliK-12 Chromosome

Cited by 22 publications

References 15 publications

Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation

Development of a Double-Crossover Markerless Gene Deletion System in Bifidobacterium longum: Functional Analysis of the α-Galactosidase Gene for Raffinose Assimilation

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

YdfH Identified as a Repressor ofrspAby the Use of Reduced GenomeEscherichia coliMGF-01

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