Gene Overexpression in Streptomyces hygroscopicus Associated with DNA Amplification

Abstract

The genetics of the Streptomyces hygroscopicus strain 10-22 is of interest due to the ability of this strain to produce antifungal compounds. Strain T110 was obtained through insertional mutagenesis of strain 10-22 and was found to have undergone DNA amplification, as determined by both conventional and pulsed-field gel electrophoresis (PFGE). pIJ702, the vector used for insertional mutagenesis, was shown to have integrated into and co-amplified with the chromosomal DNA sequence of T110, as pIJ702 hybridized predominantly with two of the three amplified BamHI fragments. The amplified DNA sequence in T110 is 10.8 kb in length and consists of 5.18 kb of Streptomyces chromosomal DNA and the entire 5.62 kb pIJ702 sequence. Sequence analysis of the 5.18 kb chromosomal sequence revealed two open reading frames, one encoding a putative IS5 family transposase and the other encoding a putative dihydroxy-acid dehydratase. Real-time PCR analysis showed that expression of the putative dehydratase gene in T110 is about 50 times greater than in the wild-type strain, consistent with the high level of amplification of this DNA region, and therefore this system has the potential for producing economically or clinically important molecules.