Duplication of biosynthetic genes to increase the yield of corresponding secondary metabolite is a practicable and successful approach. The introduction of cosmid pML48 containing partial compactin gene cluster into Penicillium citrinum 41520 enhanced compactin production (Abe et al., 2002). A large increase in nikkomycin production was obtained when an extra nikkomycin biosynthetic gene cluster was integrated into the genomic of Saccharopolyspora ansochromogenes (Liao et al., 2010). Partial duplication of
the moenomycin cluster in Saccharopolyspora ghanaensis also increased average moenomycin production (Makitrynskyy et al., 2010). In these cases, constructing and screening Selleck PR-171 the BAC or cosmid library was the routine method for obtaining
the biosynthetic gene cluster, which is time- Wnt assay and labor-consuming. In our study, the strategy of direct cloning based on Red/ET technology was applied to obtain the spinosyn biosynthetic gene cluster from the genomic DNA of S. spinosa, which is simple and convenient. This straightforward technique is particularly suitable for large DNA molecules and is therefore ideal for engineering PKS and non-ribosomal peptide synthetase pathways. The spinosyn-producing microorganism, S. spinosa, has been shown to be recalcitrant to genetic manipulation and gene transfer processes (Matsushima et al., 1994). A plasmid containing a large fragment of S. spinosa DNA can integrate at high frequencies into the S. spinosa chromosome apparently by homologous recombination, whereas a plasmid containing a small sequence (c. 2 kb) of
S. spinosa DNA integrated at low frequencies into the S. spinosa chromosome at one of two bacteriophage φC31 attB sites (Matsushima et al., 1994). Our previous Thiamet G experiments also showed low frequencies when the integrative vector pSET152 was used for conjugation from E. coli S17-1 to S. spinosa. Therefore, we only amplified the pUC replication origin, apramycin resistance gene, and oriT of RK2 from this plasmid as the linear cloning vector. The c. 18-kb spinosyn genes in plasmid pUCAmT-spn served as the homologous sequence and guided a single-crossover homologous recombination to generate stable, apramycin-resistant exconjugants with all the genes duplicated. HPLC results showed that the yield of spinosyns A and D was significantly greater in the exconjugants than in the parental strain. The exconjugants also produced three more substances which might be the minor spinosyn components. As previously described, during the early part of a spinosyn fermentation, S.