The Breeding Of High-yield Strains From Strptomyces Cavourensis TJ430

In this reserach, we obtained a Streptomyces strains which numbers TJ430 as original strain, the strain can produce an antibiotic like macrolides antibiotics, nobody reported the antibiotics. Greenhouse of the antibiotic biological activity tests showed that the aqueous solution has significant inhibitory effect for a variety of bacteria such as Phytophthora infestans, Pseudomonas corrugata Roberts et Scarlett, Sclerotinia sclerotiorum and Colletotrichum orbiculare, The control effect were 100%, 79.36%, 98.11% and 75.79%,respectively. Eventually come to the conclusion that strains TJ430 produce antibiotics has wide antibacterial spectrum and antibacterial activity, and it have a good development foreground huge.The original strain have less antibiotic production which can not meeting the demand of industrial production. The research adopted the ribosome engineering technology on the strain mutagenesis, in order to increase production of antibiotics.First we build high-yield strains screening model. The research used the 24 hole cell culture plate as a screen plate. We studied its feasibility.The results showed that affect of the antibiotic production with the Documents is negligible, the difference coefficient can controlled at 20%.The optimal time of antibiotics growing in the screening model 7 days. The production extraction is highest when using 4:1 methanol water and leaching 2 min. The above data showed that using 24 orifice plate as antibiotics screening model is feasible.This model can rapid screening of high yield strains, with time, filtering speed.Second, this experiment adopted the five factors four levels orthogonal test to research the optimization of fermentation medium. The experiments used glucose as carbon source, peptone, yeast powder, soybean meal as nitrogen source, and CaCO3 as inorganic salts, according to five kinds of materials of poor value trend graph to obtain the optimized fermentation formula for: glucose, peptone 0.6%, 1.9% soybean meal 0.3%, yeast powder 0.15%, CaCO30.015 %. We added 0.05% PVA in the fermentation process which can significantly increase the yield. And the research confirmed that the optimum cultivation time is 4 days, the initial pH is 8.5, the culture temperature is 28 ℃, with 5% inoculated quantity, and the speed is 200 RPM.The final optimization formula output 5 times higher than the original fermentation culture medium.Third, the research used the ribosome engineering for the strain screening. In our research, we used Streptomycin rifampicin and gentamicin as mutagen strains for mutagenesis, respectively. After four rounds of mutation we obtained 956 single colony. After first screening in the modle, we got 45 positive strains, 1 negative strain mutant strains. The mutation rate is about 4.7%, which celebrates the gen-136, rif-147,rif-235, rif-297, rif-297. Which antibiotics yield up to 300%, and the gen-138 have reached to 400% above. After second screening, there have 9 strains remain stable, including 8 positive mutant strain, 1 negative mutated strain. The positive mutant strains mutation rate is about 1.5 times that of the original starting strain, and the negative mutant strain was reduced by 61.8% of the original strain yield antibiotics yield.Then we extracted the rpsL gene from the gene of 9 strains, and PCR amplification and gene sequencing, we found that gene mutations do not happen in mutant strain rpsL, meaning that the ribosomal protein S12 did not change. But there have gene mutations in rpsL gene of the negative mutant strain. Speculation is mutant strain could be due to genetic mutations located in the lateral tested gene sequences, and the mutations affect the structure of the ribosome.Or it because the introduction of antibiotic resistance to activate the silence genes related to the synthesis of antibiotics, transcription a new protein, regulate the secondary metabolism of the strain, causing a high yield antibiotics. Due to the limited condition, we do not analyzed the protein. After further research, we will test the ribosome structure and protein, in order to discover new high-yielding mechanism.Fourth, we studied the experiment of strain antibacterial mechanism from the molecular level studied. We extracted the rpsL gene from the gene of 9 strains, and PCR amplification and gene sequencing, we found that gene mutations don,t not happen in mutant strain rpsL, meaning that the ribosomal protein S12 did not change. But there have gene mutations in rpsL gene of the negative mutant strain. The mutations is in 280 th ropB. Speculation is mutant strain could be due to genetic mutations located in the lateral tested gene sequences, and the mutations affect the structure of the ribosome.Or it because the introduction of antibiotic resistance to activate the silence genes related to the synthesis of antibiotics, transcription a new protein, regulate the secondary metabolism of the strain, causing a high yield antibiotics.Due to the limited condition, we have not analyzed the protein.The research adopted ribosome engineering technology to obtain the strains, the obtain mutant strains had improved significantly. The result showed that using the ribosome engineering technology to induce the antibiotic production of low microbial strains, transform second metabolic function. It is of great significance for explore the microbial strains resources, discover new active compounds.