, 2001a). Furthermore, it was formerly shown that a two- to fourfold increase in spinosad yield was attained through duplication

of the rhamnose biosynthetic genes gtt and gdh simultaneously, which implied that an extra copy of the genes involved in the conversion of the cyclized polyketide to spinosyn could enhance the spinosyn yield (Madduri et al., 2001b). Thus, we hypothesized that the overexpression of the genes for cross-bridging of the polyketide, for deoxysugar biosynthesis, attachment, and methylation could increase the flux through the pathway and accelerate the conversion of cyclized polyketide to spinosyns. The quicken see more conversion could lead Rapamycin in vitro to a faster deprivation of the cyclized polyketide and thus might stimulate the strain to synthesize more cyclized polyketide for conversion into fully glycosylated spinosyns. Interestingly, the introduction of the cosmid pRHB9A6 containing all the genes on the spinosyn gene cluster but not the PKS genes in S. spinosa A83543.3 accelerated the conversion of the PSAs, but did not enhance the spinosyn production (Madduri et al., 2001a). This may be due to the different genetic background between the two parental strains, or the negative effect of ORF-L15 and ORF-L16. The culture conditions

could also be a cause. In a more recently published report (Pan et al., 2011), the gtt and gdh genes were further overexpressed by PermE* promoter in S. spinosa SIPI-A2090, leading to a 3.81-fold

increase in spinosad production. In our study, enough a 3.88-fold increase was achieved merely by duplicating the 14 genes involved in the conversion of the cyclized polyketide to spinosyns, which indicated that an overall rise in the expression level of the spinosyn biosynthetic genes could be more effective in enhancing the spinosyn production, and our exconjugants have the potential for further improvements in production. As the Red/ET recombination is more convenient in genetic manipulating, and the integration frequency augments with the length of the homologous sequence, our strategy may be simpler and possess higher frequencies. The corresponding antibiotic might be indispensable during the fermentation of genetically engineered high-producing strains which contain a self-replicating plasmid or cosmid. In this work, the pUCAmT-spn was integrated into the chromosome by single-crossover homologous recombination, and the insertion of cloned DNA into the chromosome did not cause observable instability or loss of product yield in most cases, which provided an alternative to obtain a stable engineered strain. In conclusion, the Red/ET approach was applied to clone part of the spinosyn biosynthetic gene cluster from the genomic DNA of S. spinosa, allowing the generation of a stable spinosyn overproducer.