Construction Of Saccharopolyspora Spinosa S04-41 Disrupted Of RelA And Its Effects On Mutant Strain

Spinosyn is a new biological pesticides, which can control most variety of pests, including lepidopterans, orthopter, diptera and coleopterans. Spinosyns are secondary metabolites of soilactinomycete, Saccharopolyspora spinosa, through aerobic fermentation. The most active of the spinosyns are spinosyn A and D, named spinosad. Spinosyns has great prospects with a broad spectrum insecticide, insecticidal activity, and environment-friendly characteristics.However, the spinosyns-producing capability of wild type of Saccharopolyspora spinosa is very limited, and the researchers have to focus on the strain improvement, strategy of traditional mutation is changing gradually to the directional genetic method used matabolic engineering principles. This reaserch for the purpose of increasing production of spinosyn, the Saccharopolyspora spinosa mutant type strain disrupted of relA was constructed. The gene relA codens RelA protein, which catalyze (p)ppGpp. (p)ppGpp can mediate bacterial stringent response to environment stress including nutritional starvation. (p)ppGpp also as a globle factor regulates genetic replication, transduction, and expression in the strains of circulating to change from the logarithm to stationary phase, widely inhibits bacteria growth-related gene expression, and promotes the stable-related gene expression, therefore affects the synthesis of secondary metabolites type, production and synthesis time. The 1.15kb length part of relA segment was amplified used Saccharopolyspora spinosa S04-41 genome DNA as template, and was digested with EcoR I/Hind Ⅲ, was further connected to pOJ260, as a result the recombinational plasimid named pOJ260-relA was constructed. Futher, the recombination carrier pOJ260-relA was electrotransfer into E.coli S17-1, and transfered into S. spinosa S04-41 by conjugational transfer, and intergrated into the chromosome through the homologous recombination. The conjugant mutant strain was confirmed with PCR amplification, and the results showed that relA was successfully desturbed in Saccharopolyspora spinosa. The mutant strain was named S. spinosa SP-relA which has excellent stability. Compared with wiled strain S. spinosa S04-41, the spinosynA and spinosynD production of S. spinosa SP-relA has respectively improved 1.84 and 1.43 times, can produce a red-brown pigment, advance into stationary phase with faster growth speed. However, spinosa SP-relA growth trend is consistent with wild strain S. spinosa S04-41, and there is no obvious change in the term of morphology.In conclusion, this research provide some valuable information for further improving spinosyn-producing in S. spinosa, through up the positive regulator and down the negative regulator of pathway-specific regulation, increasing precursor-supplying by overexpressing key precusor biosynthesis enzymes, and reducing spinosyns production except spinosad around spinosyn biosynthetic pathway.