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

Abstract

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 It has had two strains sequenced: R (3,314,179 bp [our unpublished data]) and ATCC 13032 (3,309,401 bp [14] or 3,282,708 bp [17]).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...

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FurC Regulates Expression of zupT for the Central Zinc Importer ZupT of Cupriavidus metallidurans

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