SUMMARY: We previously reported the cloning of a 1.6 kb III fragment (containing the junction of the repeating unit) from chromosomal DNA of strain B7 in which tandem amplification of a 16 kb region occurred, and the induction of B7-type gene amplification by competence transformation with this cloned fragment. Based on this result, we designed, on the chromosome, a gene amplification of the 22 kb repeating unit containing the α-amylase structural gene (), the tunicamycin-resistance gene () and the shikimate kinase structural gene (). We cloned only two short DNA fragments from both termini of the 22 kb region, constructed a junction structure of the designed repeating unit on pBR327 and transformed a wild-type strain by this constructed plasmid. As a result, we succeeded in obtaining tunicamycin-resistant (Tm) transformants in which the designed gene amplification of 22 kb occurred on the chromosome. The Tm transformants showed high productivity of α-amylase and shikimate kinase. The copy number of the repeating unit was estimated to be 10-20. This system may provide an effective means of amplifying long (> 20 kb) DNA regions on the chromosome.


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