Study On The Breeding And Technology Of High Efficient Sisomicin Synthesis Strain

Sisomicin is a broad-spectrum aminoglycoside antibiotic with similar structure and antibacterial profile to gentamicin.It has antibacterial effect on Gram-negative bacteria such as Staphylococcus aureus,Escherichia coli,Proteus and Pseudomonas aeruginosa.Among them,the antibacterial effect on pseudomonas aeruginosa is stronger.Sisomicin can not only be used in clinical treatment directly,but also can be used as a precursor for the synthesis of semisynthetic antibiotics netilmicin and prazolmicin with low toxicity.A high-yielding strain of sisomicin,Micromonospora inyoensis I4-10,was obtained by protoplasma-induced mutation at Atmospheric and Room Temperature Plasma in the laboratory,and its biological titer was stabilized at about 1478.25 U/m L through fermentation optimization.However,compared with gentamicin and kanamycin,its biological titer was still low,which restricted its further production and application.Therefore,in this study,Micromonospora inyoensis I4-10 was used as the starting strain to reduce or remove the product inhibition effect during the fermentation of sisomicin by adaptive laboratory evolution（ALE）.Then there is further high efficiency synthesis of sisomicin strains through genome shuffling and rifamycin resistance screening,and optimization of fermentation technology for dominant strains,to increase the biological titer of Sisomicin.Key findings were as follows:（1）Strain breeding based on adaptive evolution in the laboratory.In order to improve the tolerance of M.inyoensis I4-10 to sisomicin,the stress pressure value of strain adaptive evolution was determined by product tolerance study,and the initial pressure value was determined as 1.6 g/L sisomicin concentration.After several rounds of laboratory adaptation evolution,the concentration of the strain adapted to sisomicin increased to 5.8 g/L after 50generations,and two high-yielding strains A20-10 and A50-12 with higher tolerance to sisomicin were finally screened.The fermentation results showed that the fermentation cycle of A20-10 was shorter,and the biological titer of sisomicin reached 1635.69 U/m L at 120 h.The biological titer of sisomicin in A50-12 was higher,reaching 1846.06 U/m L at 144 h,which was 24.9%higher than that of the original strain（1478.25 U/m L）.（2）Resistance screening breeding based on genome shuffling technology.Strain A20-10 with the advantage of short fermentation cycle and strain A50-12 with the advantage of high biological titer were used as parent strains for genome shuffling.Firstly,the inactivation conditions of parents,the concentration of resistance screening and the protoplast fusion conditions were investigated respectively.The heat inactivation conditions were determined as1 min at 70℃,UV inactivation conditions as 200 s,and resistance screening conditions as rifamycin 10μg/m L.The optimal fusion time and solvent promoting PEG4000 concentration were 10 min and 40%,respectively.Through two rounds of genome shuffling breeding,a high-yielding strain GS2-137 was selected from 300 mutant strains,and its biological titer of sisomicin was increased to 2036.50 U/m L,10.3%higher than that of A50-12.Meanwhile,the fermentation performance of GS2-137 was relatively stable,and the biological titer of 10successive passages was stable at about 2000 U/m L.（3）Optimization of fermentation process of high-yielding strain GS2-137.The optimal seed medium for high yield strain GS2-137 was:soluble starch 10 g/L,Sucrose 10 g/L,Peptone5 g/L,Yeast powder 5 g/L,K₃PO₄·3H₂O 2 g/L,Mg SO₄·7H₂O 0.5 g/L,Ca CO₃ 3 g/L.The best age for 24 h,best inoculation amount to 5%.And components of fermentation medium in the most important factor of influence on sisomicin synthetic kinds and concentrations of nitrogen source is optimized.The results showed that the combination of three nitrogen sources（soybean cake powder,beef powder,sodium nitrate）was superior to two nitrogen sources（soybean cake powder,beef powder）and single nitrogen source（soybean cake powder）.When 30 g/L soybean cake powder,6 g/L beef powder and 1 g/L sodium nitrate were added as nitrogen source,the biological titer of sisomicin reached 2182.33 U/m L.On the basis of the above work,the effects of p H and dissolved oxygen on the fermentation of Sisomicin by high biological titer strain GS2-137 were investigated at the level of 5 L fermenter.By regulating p H7.3 and 30%dissolved oxygen,the biological titer of Sisomicin was 1430.03 U/m L.