Isolation and Characterization of a Native Composite Transposon Tn14751 Carrying 17.4 Kilobases of Corynebacterium glutamicum Chromosomal DNA

摘要

A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 × 10² transformants per μg of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.