A new insertion sequence, IS14999, from Corynebacterium glutamicum

Tsuge, Yota; Ninomiya, Kana; Suzuki, Nobuaki; Inui, Masayuki; Yukawa, Hideaki

doi:10.1099/mic.0.27567-0

Cited by 10 publications

(9 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, despite its industrial usefulness, similarly useful techniques have yet to be developed for C. glutamicum. In our laboratory, comprehensive studies on C. glutamicum, including isolation and characterization of new transposable elements and development of Cre/ loxP-mediated genome deletion systems, are being undertaken (7,16,32,33,35,38). The utilization of such techniques could greatly contribute to creation of a C. glutamicum-based MGF.…”

mentioning

confidence: 99%

New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

Suzuki

Nonaka

Tsuge

et al. 2005

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.The whole-genome sequences of more than 200 organisms have been deciphered since the first genome sequence was determined in 1995. These genome sequences have become an important resource for a more comprehensive understanding of cellular life. The availability of whole-genome sequences allows us to decipher the roles of thousands of genes. Corynebacterium glutamicum is a well-known industrial strain widely used for the production of amino acids, nucleic acids, and organic acids (18,23). It has had two strains sequenced: R (3,314,179 bp [our unpublished data]) and ATCC 13032 (3,309,401 bp [14] or 3,282,708 bp [17]). Based on whole-genome sequences, strain reconstruction studies for improved industrial applications have been initiated (28). In the field of bacterial genomics and metabolic engineering in the postgenome era, the concept of minimum genome factories (MGFs) has been proposed (16). These can be defined as recombinant strains whose metabolism has been streamlined to the optimal minimal subset in order to maximize product formation for targeted applications. C. glutamicum is one of the most widely used bacteria for bioindustry, and the improvement of its genome is important for enhanced production of biochemicals.To implement the concept of MGFs by the rearrangement of bacterial genomes, molecular biology tools that make multiple excisions and insertions possible are a prerequisite. For Escherichia coli, the development of genomic engineering techniques utilizing bacteriophage recombinases, homologous recombination, or transposable elements has been reported recently (6,10,12,20,39). By use of such techniques, the construction of a deletion mutant whose genome size was reduced by 1.38 Mb was reported (12). However, despite its industrial usefulness, similarly us...

show abstract

mentioning

confidence: 99%

New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

Suzuki

Nonaka

Tsuge

et al. 2005

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…The basic strategy for southern hybridization was described previously. 18) A probe was prepared with PCR using 13655FP and 13655RP. Genomic DNA was extracted and digested with EcoRI, which does not cut within IS13655.…”

Section: Methodsmentioning

confidence: 99%

“…The transformation was performed as described previously. 18) All plasmid DNA used in the transformation of C. glutamicum was extracted from E. coli JM110 (dam À dcm À ). One microgram of unmethylated plasmid was used to transform C. glutamicum cells using a GenePulser II (BioRad, CA), as previously described.…”

Section: Methodsmentioning

confidence: 99%

“…[11][12][13] In C. glutamicum, some functional transposable elements have been identified, including IS31831, IS1206, IS1207, Tn14751, and IS14999. [14][15][16][17][18] In this study, we isolated and characterized a new functional IS3 family element, IS13655, from the C. glutamicum ATCC13655 strain. The transposon vectors of this IS element was constructed, and then the transposition efficiency and transposition sites were determined.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Isolation of a New Insertion Sequence, IS13655, and Its Application toCorynebacterium glutamicumGenome Mutagenesis

Tsuge

Suzuki

Ninomiya

et al. 2007

Bioscience, Biotechnology, and Biochemistry

Self Cite

View full text Add to dashboard Cite

A new functional Corynebacterium glutamicum insertion sequence (IS) element, IS13655, was isolated using a suicide vector. The IS element was 1,293 bp in size and contained 26-bp imperfect inverted repeats (IRs) and 3-bp target site duplication as direct repeats (DRs). IS13655 harbored two ORFs with high similarity to the transposase of IS1206, an IS3 family element. IS13655 revealed relatively high transposition efficiency, with low target site selectivity along the Corynebacterium glutamicum R genome, making it a potentially useful genetic engineering tool.

show abstract

“…The presence of mobile elements from the IS3 family, which is characterized by two consecutive and partially overlapping open reading frames, was observed in the chromosome of C. glutamicum (IS1206) (14) and on native plasmids of Corynebacterium jeikeium (119). IS14999, isolated from C. glutamicum (131), represents the only member of the IS630/Tc1 mobile element family identified to date in corynebacteria. This particular mobile genetic element is more closely related to eukaryotic Tc1/mariner elements, known not to require any host factor for transposition, than to prokaryotic elements.…”

Section: Isolation Of Native Mobile Genetic Elementsmentioning

confidence: 99%

Manipulating Corynebacteria, from Individual Genes to Chromosomes

Vertès

Inui

Yukawa

2005

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Corynebacterium glutamicum is an industrial organism with a long history of use for the production of various fine chemicals. The C. glutamicum-mediated production of L-glutamic acid by fermentation was one of the very first industrial processes of the biotechnology era. This fermentation originated in Japan from the discovery in 1957 by Kinoshita et al. (cited by Kumagai [68]) that, under suitable conditions, this soil bacterium is able to secrete L-glutamic acid in significant amounts (66). This process successfully replaced less cost-effective methods based on hydrolysis by concentrated hydrochloric acid of soy or various other plant proteins. Glutamic acid was traditionally extracted from seaweed to serve as a seasoning agent and remains to this day a very important food flavor additive, the worldwide production of which approximates 1.5 million tons per year (43). Industrial glutamic acid fermentation was implemented early on by major Japanese companies, thus building over time exquisite manufacturing knowledge at various industrial scales up to approximately 5,000 hectoliters (43). In the 1970s, efficient C. glutamicum L-lysine and L-threonine producers were generated by random mutagenesis and positively selected by phenotypic resistance to lysine or threonine analogs. The advent of molecular biology enabled a new wave of development in which this industrial know-how was leveraged, not only to improve the performance of the existing lysine and threonine production processes, but also to enable the production of other amino acids and vitamins (29,43,51,68). The utilization of recombinant DNA techniques, combined with metabolic and carbon flux analyses (67, 99), facilitated the identification of metabolic bottlenecks and their bypassing by expressing or repressing the corresponding genes to develop further improved amino acid industrial production processes. The intrinsic characteristics of this food grade microbial workhorse include its lack of pathogenicity and its lack of spore-forming ability, both desirable traits as listed by the U.S. Center for Biologics Evaluation and Research and the U.S. Center for Drug Evaluation and Research guidelines, as well as its high growth rate, its relatively limited growth requirements, the ability of several strains not to undergo autolysis under conditions of repressed cell division (50), the absence of native extracellular protease secretion that makes corynebacteria suitable hosts for protein expression (10, 100), and the relative stability of the corynebacterial genome itself (85). These intrinsic attributes, combined with an up-to-date set of genetic-engineering tools, make this organism ideal for the development of robust industrial processes that are increasingly competitive compared to Escherichia coli-, Bacillus subtilis-, or yeast-based processes. As a result, corynebacterial fermentations have become increasingly relevant to a wide range of industrial sectors, including food, feed, cosmetic, pharmaceutical, and chemical companies.Nonetheless, the significance...

show abstract

A new insertion sequence, IS14999, from Corynebacterium glutamicum

Cited by 10 publications

References 39 publications

New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

Isolation of a New Insertion Sequence, IS13655, and Its Application toCorynebacterium glutamicumGenome Mutagenesis

Manipulating Corynebacteria, from Individual Genes to Chromosomes

Contact Info

Product

Resources

About