Nevertheless, some reports revealed that massive amplification

of antibiotic biosynthesis gene cluster is often one of the outcomes of empirical strain improvement programs. The kanamycin-overproducing strain, Streptomyces kanamyceticus 12-6 generated by classical mutagenesis possesses tandem amplification of the entire kanamycin (Km) biosynthetic gene cluster and the level of Km production is linearly BIBF 1120 co-related with the copy number of the Km biosynthetic gene cluster (Yanai et al., 2006). A penicillin-overproducing strain of Penicillium chrysogenum contains a large number of copies of penicillin biosynthetic genes (pcbAB, pcbC, and penDE) in tandem on a c. 57.9-kb DNA fragment (Fierro et al., 1995). In the industrial strain Streptomyces lincolnensis 78-11; the non-adjacent gene clusters for the production of lincomycin and melanin are duplicated (Peschke et al., 1995). These examples imply that introduction of extra copies of biosynthetic gene clusters into a wild-type strain might be an effective approach to improve the yield of the corresponding product. However, most of the antibiotic biosynthetic genes often cluster in a contiguous region containing tens of thousands of nucleotide base pairs in the chromosome. They are consequently almost impossible to manipulate via restriction endonucleases

and DNA ligases due to the frequent occurrence of cleavage sites. Reports focusing on overexpressing these biosynthetic gene clusters in parental strain through directed genetic approaches are few. The Red/ET recombination ABT-199 nmr technology provides a convenient and simple method for engineering large DNA fragments in Escherichia coli. The recombineering is mediated by homologous recombination, which occurs between two DNA molecules and requires only short homology regions (c. 40–50 bp) for efficient recombination (Zhang et al., 2000). The myxochromide S (mchS, c. 30 kb) and myxothiazol (mta, c. 60 kb) gene clusters from the myxobacteria

Stigmatella aurantiaca, the epothilone (epo, c. 60 kb) gene cluster from myxobacteria Sorangium cellulosum have all been successfully engineered for heterologous expression by different strategies based on Red/ET technology (Wenzel et al., 2005; Perlova et al., 2006). The S. spinosa CCTCC M206084 isolated by our laboratory has a low capability for spinosyn production. We thus attempted Pregnenolone to improve its spinosyn productivity through duplication of the spinosyn biosynthetic genes. It is difficult to obtain the c. 74-kb gene cluster on one single vector by one step and therefore we first directly cloned part of the spinosyn biosynthetic gene cluster (c. 18 kb) which encoded the enzymes for cross-bridging of the cyclized polyketide, for deoxysugar biosynthesis, attachment and methylation from the genomic DNA of S. spinosa CCTCC M206084 with the assistance of Red/ET recombination instead of constructing a genomic library. The resultant plasmid pUCAmT-spn was then introduced into S. spinosa CCTCC M206084 through conjugal transfer.