Strain Improvement Of Streptomyces Spectabilis And Optimization Of Culture Medium For Spectinomycin Production

In this study, the genome shuffling combined the traditional mutagenesis was applied to improve Streptomyces spectabilis which produced spectinomycin. Response Surface Methodology （RSM） was adapted to optimize fermentation medium for spectinoymcin production, which was verified for scale-up processes in 5 L fermentor.Four strains UV-9, UV-12, UV-18, and UV-21 were obtained with spectinomycin production increasment by 108.7%, 107.5%, 109.9% and 107.1% respectively comparing with the initial strain after UV irradiation process on protoplasts of initial strain Streptomyces spectabilis XZ-4. Then, two rounds of protoplast fusion were carried out by PEG among the four strains and one fusant strain BR2-1 was screened out with production of spectinomycin increasing by 46.6% comparing with that of UV-18, and by 207.7% comparing with that of origin strain. The protoplast recombinants were identified by SDS-PAGE electrophoresis of their solvent protein, which showed that the recombinant strains gave more protein bands than origin strain.NTG was adapted to mutate strain BR2-1. Two mutants with Neomycin-resistant X40-2, X40-5 and two mutants with streptomycin-resistant stains L30-1, L50-1 were screened out for much increasment of spectinomycin production, in which L30-1 had the highest increasment by 30%. The protoplasts from these four strains were fused equally, and then regenerated on regenerating media plate including streptomycin 30 U/mL and Neomycin 20 U/mL. The fusant plates were again subjected to protoplast fusion and then regenerated, isolated and selected for evaluation for spectinomycin production. One double resistant strain LXR1H-8 was obtained with high production of spectinomycin which increased by 56% and 486.3% compared with its parent strain L30-1 and the origin strain, respectively.The optimization of culture medium was carried out with Response Surface Methodology （RSM） on carbon source, nitrogen source and inion Co2+ for spectinomycin production by Streptomyces spectabilis YZ1-6. The result showed that the strain gave high spectinomycin production, which added up to 7769.8 U/mL and increased by 261.7% compared with the origin medium. Also, the effects of microelementon, amino acid, vitamine and divalent inion on the synthesis of spectinomycin was studied, the result showed that Mn²⁺, Cu²⁺, Co²⁺had positive effects on spectinomycin production, while glycin, arginine, methionine, glutamine, aspartic acid, vitamine B6, vitamine B12 had negative effects.The study on the quality of the seed for scale up fermentation process showed that the best seed was at age of 36 h （with pH 7.3, residue sugar 0.38%, biomass of 6.87 g/L in culture broth）. With the cultures as seed, the strain gave high spectinomycin production on 5 L fermentation of 4800 U/L.