Preliminary Application Of Ribosome Engineering Technology In Saccharopolyspora Spinosa

Spinosad is a kind of secondary metabolite of aerobic fermentation of Saccharopolyspora spinosa, with biological characteristics of high insecticidal activity, no damage to non-targets and unusual mechanism of killing pests and so on. It has a wide market prospects in biocontrol of insects in agriculture and grain-store. According to ribosome engineering, we applied chemoresistance mutation method to screen Spinosad-like products of S. spinosa. So far, ribosome engineering was first used in S. spinosa, which means that ribosome engineering not only laid research foundation for its application in other kinds of microbes, but created a new and effective pathway to screen stains with highly productive Spinosad. Our work obtained here was as follows:1. S. spinosa growth was inhibited in the medium containing 40μg/mL Streptomyces, indicating the minimal inhibitory concentration (MIC) was 40μg/mL.2. We had established a continuous resistance screening method. And two new components different form Spinosad A and D were detected from a Streptomyces resistence mutant of S. spinosa wtih HPLC.3. To purify one of new components, we had used kinds of methods, including macroporous resin, silica gel chromatography and preparative chromatography, and a component in white powder (99.6% purity quotient) was obtained.4. The structure of this new component was further identified with IR, NMR, mass spectrometry. The new structure was C33H50O9 with a molecular weigh of 590.74. It was different from Spinosad in that it was lack of forosamine. This component might have different insecticidal activity, biological synthesis mechanism, and metabolic pathways.5. Through analysis of DNA sequence, we found that rpsL gene of the Streptomyces resistance mutat of S. spinosa had two point mutations (A128G and G341A) in ribosome Streptomyces sensitive domain. Since rpsL gene can improve translation activity of proteins by increasing translational elements, it can activate the regulation of secondary metabolism. Whether the point mutations were related with the two new components still remains unknown.