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Preliminary Application of Ribosome Engineering Technology in Saccharopolyspora Spinosa

Author: ChenLiXian
Tutor: GuanXiong；HuangTianPei；ZhangXiaoLin
School: Fujian Agriculture and Forestry University
Course: Biochemistry and Molecular Biology
Keywords: Spinosad The thorn sugar and more sp New substances Separation and purification Structural identification
CLC: S482.3
Type: Master's thesis
Year: 2010
Downloads: 86
Quote: 0
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Abstract


Spinosad, also known as multi-kill adriamycin (of Spinosad) is produced in the cells of secondary metabolites produced by the aerobic fermentation of the barbed Saccharopolyspora bacteria (Saccharopolyspora spinosa), with high insecticidal activity, non-target animals does not work and insecticidal mechanism unusual biological characteristics, on the prevention and control of field pests and stored grain pest has broad market prospects and a good value. According to ribosome engineering theory, the use of the drug resistance mutation Screening spinosad overproducing strain, which is the ribosome engineering technology for the first time in Saccharopolyspora genus in the application, not only for ribosomal technology in the future in other species of microorganisms The application has laid a foundation, and opened up a new and effective way spinosad PRODUCING STRAIN. This article by ribosome engineering Mutation Breeding spinosad mutants, the main conclusions are as follows: 1, the determination of the minimum inhibitory concentration (MIC). In the trial found the barbed Saccharopolyspora sp of the Streptomyces-containing 40μg/mL when growth suppressed, so the minimum inhibitory concentration of 40μg/mL. 2, to establish a continuous Resistance Screening culture method, and a streptomycin resistant mutant of fermentation product was found by high-performance liquid chromatographic analysis method wherein there are two spinosyns components A and D is not the same as the group minutes. 3, the separation and purification of new substances. Through with macroporous resins, silica gel chromatography and preparative chromatography, etc. methods wherein a new substance for separation and purification, to give one component content of 99.6% as a white powder. 4, the structure of the new substances identified. By IR, NMR, mass spectrometry for the structural identification of new substances and new substances Formula C33H50O9, a molecular weight of 590.74, compared with the the spinosad series matter structure in the quad-core ring part on the lack of Fuller amine sugar (forosamine a) component may be provided with known spinosyns series was the difference in insecticidal activity, the biosynthetic mechanism and metabolic regulation pathways. 5, barbed Saccharopolyspora bacteria resistant mutant DNA sequence analysis, found in the A 128 was mutated to G mutation from lysine (AAG) for the arginine (AGG), 341 of the G is mutated to A, by arginine (CGC) mutation leucine (CAC) for the group, these mutations in the rpsL gene (encoding ribosomal S12 protein) streptomycin sensitive domain. Known to increase the activity of protein translation through increased translation factor rpsL gene, thus activation secondary metabolic regulation. The rpsL gene point mutations thorn Saccharopolyspora bacteria streptomycin resistant mutant generated two new substances subject to further study whether there is a direct link.

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CLC: > Agricultural Sciences > Plant Protection > Pesticide ( chemical control ) > Various pesticides > Pesticides
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