S2). Fermentation broths of S. spinosa CCTCC M206084 and three exconjugants were detected by HPLC and HPLC-MS. All samples revealed two compounds with the same retention times as those of the standard spinosyn A and spinosyn D (Fig. 2). Their identities were further confirmed by HPLC-MS, showing a measured m/z of 732.6 and 747.0 (M + H)+ which were consistent with the molecular formula C41H65NO10 for spinosyn A and C42H67NO10 for spinosyn D, respectively (Fig. S3). Three exconjugants enhanced their production of spinosad ranging from 1.90- to 2.24-fold when fermented in the production medium PM1. Fermentation in the modified

production medium PM2 showed a similar trend of increased spinosad production, with S. spinosa trans1 showing the highest increase. According to the standard curve, the total concentration of spinosyns A and D of S. spinosa trans1 in production medium PM2 was 388.0 Dasatinib (± 25.0) mg L−1, which overproduced 3.88-fold spinosad compared with 100.0 (± 7.7) mg L−1 in the parental strain. Analysis of variance by spss 16.0 showed that the increases of spinosad

production in the three exconjugants when compared with that of the wild-type strain were statistically significant. Furthermore, three extra peaks were observed in the chromatogram of the mutant strain but not UK-371804 molecular weight of the wild-type strain (peaks marked with an asterisk, Fig. 2). The HPLC-MS result indicated that these peaks might have a m/z of 718.0 (M + H)+. As the spinosyns B, E, F, H, J, and K all had a relative molecular mass of 718.0 (Sparks et al., 2008), we speculated that they could be minor spinosyn derivatives. The exconjugants and the wild-type strain shared a comparable final biomass

(data not shown), implying that higher biomass was not an overproduction mechanism. Saccharopolyspora spinosa trans1, which had the highest increase in spinosad production among the three exconjugants, was chosen to further assess gene dose effect on increasing the enzyme production. According to the time course for spinosad production of the parental strain and S. spinosa trans1 in production medium PM1 (Fig. S4), the total RNA was extracted from the fifth day fermentation culture for RT-PCR analysis. spnK, spnI and spnH were selected from three different transcript units. The transcript level of the gene fragment PtdIns(3,4)P2 of sigA served as a control in this study. The transcript levels of spnK, spnH, and spnI in recombinant strain trans1 were 3.203-, 3.524- and 3.495-fold higher than those in the parental strain, respectively (Fig. 3). The increase in transcript levels for spnK, spnI, and spnH agreed with the high yield of spinosad in the exconjugants. Exconjugants of S. spinosa CCTCC M206084 were passaged in the absence of selection in TSB for 16 culture doublings (Matsushima et al., 1994), and then plated on brain heart infusion broth (BHI; Difco) with Am (50 μg mL−1) and without Am.