Neomycin is a kind of aminoglycoside antibiotic,is a secondary metabolite produced by the fermentation of Streptomyces fradiae,having strong inhibitory effect on some Gram-positive bacteria and Gram-negative bacteria.Neomycin is often used as an external preparation to treat skin infectious diseases,such as eye drops,tinctures,creams,etc.The demand for neomycin sulfate-related products with huge development potential in the world market is growing rapidly,so it is of great significance to use genetic engineering technology to improve the level of neomycin fermentation.Most of the neomycin production strains currently used in China are selected by mutagenesis.However,due to the fact that the strain is prone to degradation and the fermentation process is lagging behind,the domestic production level of neomycin is generally low,so it is of great significance to use genetic engineering technology to improve the fermentation level of neomycin.This study firstly improved the microbial assay of neomycin.The test organism of a pathogenic bacteria Staphylococcus aureus was replaced by the Bacillus subtilis(spore),which was generally recognized as safe(GRAS).The substitute strain was also sensitive to neomycin sulphate and a calibration curve between the standard neomycin sulphate concentration and diameter of B.subtilis inhibition zone was constructed.B.subtilis(spore)instead of S.aureus as a test organism not only increases the operation safety,but also simplifies the actual operation procedues.It is a simple and efficient neomycin determination method,worthy of further application.In this study,a new strain of neomycin-producing strain Streptomyces fradiae ATCC 10745 from the American Type Culture Collection(ATCC)was systematically selected and researched.First,through natural selection of strains,agar block screening As well as shake flask fermentation verification and other means,the strain was screened and rescreened,and an excellent strain XG3-1,which is fast growing and multiplying,high in production,high genetic stability,suitable for industrial production of neomycin.The neomycin potency can reach 22010 U/mL.After 5 consecutive passages,the strain can still maintain a high production level of neomycin without obvious degradation.On this basis,a complete set of genetic operation system was established for this strain.Through molecular biology and other means,a genetic modification method using thiostrepton as a selection marker was successfully identified,and the resistance marker gene(tsr ORF)was integrated into a specific site in the strain’s genome,which can achieve the stability of Streptomyces fradiae.Directional transformation.Four enzymes(neo6,neo 7,neo17,neo18)related to the synthesis of 2-DOS,the attachment of ribose,the formation and attachment of aminohexose in the neomycin biosynthesis gene cluster were selected,and their genes were blocked,The results show that after the gene blockage,the four strains no longer produce neomycin,which proves that these four genes are the key enzyme genes involved in neomycin biosynthesis pathway.Then over-expressed these four key enzyme genes separately,the results show that the over-expression of the 2-DOI synthase gene(neo7)in the first step of the neomycin biosynthesis pathway can increase the yield of neomycin by 21.9%,up to 26840 U/mL.Using the obtained high-yield strains to optimize its fermentation process,six factors including glucose,rice flour,ammonium sulfate,peptone,yeast powder and peanut cake powder were selected as the investigation objects,and L25(56)orthogonal design method was used to optimize the neomycin fermentation medium.After optimization,the neomycin titer could reach 27972 U/mL,which is 4.2%higher than the original fermentation medium. |