Mutagenesis Breeding And Fermentation Optimization Of VGM-producing Streptomyces Virginiae

Virginiamycin(abbreviated as VGM)is a chain-positive antibiotic produced by the fermentation of Streptomyces virginiae and consists of various components which mainly include Mi factor of macrolipids and S,factor of cyclic polypeptides.Because of its novel structure,safety,non-toxicity,and low resistance to mutant strains,VGM is a rare animal feed additive with promising applications.At present,China has been imported a large amount of this product each year,and its cost is relatively high.Therefore,it is of great social and economic benefits for mutagenesis and breeding of Streptomyces virginiae and optimization of fermentation processes.This project first established a high-throughput screening method that uses 24-well plate as a miniature culture platform for mutagenizing strains and combined with tube dish method and high-performance liquid chromatography(HPLC)for detection and screening of virginiamycin in order to screen high-yield VGM mutants.The tube dish method is simple,convenient and easy to observe and mainly used for the preliminary screening of a number of mutagenizing strains and can rapidly increase the screening efficiency.The HPLC detection method is the most accurate and sensitive,so it is used as a quantitative detection method for VGM.Secondly,single-factor experiments were used to determine the optimal medium for fermentation with a volume of 1.25 mL,an optimal inoculum of 5%,an optimal fermentation time of 30 h,and an optimal rotation speed of 240 rpm in a 24-microplate culture system.The use of 24-well plate microculture system for the rapid screening of a large number of strains is feasible and effective,with obvious advantages.According to the basic theory of biosynthetic metabolic pathways and metabolic regulation and breeding,this study used Streptomyces virginiae FL3 as the starting strain,breed by ultraviolet light,microwave,sodium nitrite and other mutagenesis factors and precursor and Streptomycin sulfate as a resistance screening agent,so that the ability of Streptomyces virginiae to produce VGM is continuously improved,and finally a genetically stable streptomycin-resistant strain MWS178 was selected.The fermentation unit of this strain is 146.72 ug/mL,which is about 6 times higher than that of the starting strain.At the same time,it was found that the positive mutation rate of microwave mutagenesis based on streptomycin sulfate was as high as 32.83%,and mutant strains with high yield of VGM were more easily obtained.In this study,the fermentation medium and culture conditions of Streptomyces virginiae MWS178 were optimized.The effects of inoculum size,carbon source,and nitrogen source on fermentation were investigated through single factor experiments,and the best fermentation medium was obtained(/L):glucose 10,soluble starch 10,soybean meal 20,yeast extract 10,NaCl 2.5,pH 7.the best culture conditions of shake flask fermentation:species age 16h,inoculum volume 5%,liquid volume 30mL/250mL flask,temperature 28 ℃,fermentation 48h.Based on the above optimized fermentation medium and culture conditions,the final production VGM of strain MWS178 was as high as 460.23 μg/mL,which was more than three times that of the original fermentation process.In recent years,reports on the influence of rare earth elements on microbial fermentation have been increasing year by year.This subject further investigated the effects of six rare earth elements on VGM production for the first time.The results showed that different rare earth elements had different effects on VGM yield,including 200 mg/L cerium chloride,300 mg/L samarium trinitrate,3 mg/L strontium chloride and 10 mg/L lanthanum chloride before fermentation.The VGM production respectively increased by 13.13%,22.64%,23.91%,and 32.60%,compared with the control.Further study found that the best addition time of lanthanum chloride was the initial stage of fermentation.Adding 10 mg/L lanthanum chloride at the beginning of fermentation,the yield of strain MWS178 was as high as 610.28 ug/mL,which was about 28 times higher than that of the original strain FL3.