【作者】 张海霞；

【导师】 陈少欣；

【作者基本信息】 中国医药工业研究总院 ， 微生物与生化药学， 2021， 硕士

【摘要】 交沙霉素属于大环内酯类抗生素。结构与麦迪霉素相似,抗菌谱和红霉素相似。临床上在治疗急性呼吸道感染和泌尿道支原体感染方面效果显著,在口腔感染的治疗方面效果也很好,目前主要通过发酵获得。国内的原料药均是进口而来,因此,提高交沙霉素的发酵单位,在国内实现工业化生产具有重要意义。本文通过建立交沙霉素抗性筛选模型,结合传统紫外诱变、60Co-γ诱变、DMS诱变、NTG诱变,从出发菌株那波链霉菌(Streptomyces narbonensis)J-1-13(360.3 mg/L)开始经过多次的诱变选育,得到菌株J-7-43。与菌株J-1-13相比,其发酵单位提高了 2.4倍,达到了 851.2 mg/L。在此基础上通过培养基以及培养条件的优化,确定了最佳的发酵配方,即碳源为2%玉米淀粉和8%菜籽油、氮源为3%棉籽饼粉和1.5%谷朊粉、异戊醇为0.2%;最佳的培养条件,即斜面生长8 d、种龄36 h、接种量10%、发酵天数7 d、温度28℃,发酵培养基初始pH为7.0。通过以上条件的优化,使菌株J-7-43的发酵单位达到 924.9 mg/L。接着在摇瓶培养的基础上,对5L发酵罐的工艺做了调整,增加初始菜籽油的比例为10%,并在发酵后期补加菜籽油维持其比例为6%,使发酵罐的单位达1253.6 mg/L。此后,在补油的基础上,添加0.2%异戊醇在发酵培养中,并从第4 d开始每天补加0.1%异戊醇,使得交沙霉素的发酵单位达到1586.3 mg/L,且明显降低了4"-O-丁酰-交沙霉素的比例,为后期分离纯化降低了难度。另一方面,本研究还对交沙霉素产生菌J-2-40进行了全基因组测序,利用生物信息分析推测出交沙霉素的生物合成基因簇,进而分析了交沙霉素的生物合成途径,并验证了合成基因簇上两个后修饰基因orf9和orf29,以及乙酰-CoA羧化酶编码基因accA2BE对交沙霉素产量的影响,为交沙霉素的菌种基因改造做了初步的探索。更多还原

【Abstract】 Josamycin,a macrolide antibiotics,is a member of the leucomycin family.The structure of josamycin is similar to midecamycin and leucomycin.Josamycin can be used clinically to treat acute respiratory infections and urinary tract infections,and it is also effective in oral infections.At present,it is mainly obtained by fermentation.Because all of the josamycin in China is imported from Japan,it is necessary for us to increase the production of josamycin and realize the localization of the API.In this study,the screening model of josamycin resistance was combined with conventional UV mutagenesis,60Co-γ mutagenesis,DMS mutagenesis,and NTG mutagenesis.After several mutagenesis and selection,a strain J-7-43 was obtained,which was 2.4 times higher than that of the original strain J-1-13,and the fermentation titer reached 851.2 mg/L.After that,we optimized the medium and culture conditions.The optimal carbon source was 2%corn starch and 8%rapeseed oil,and the optimal nitrogen source was 3%cottonseed cake flour and 1.5%gluten.The optimal proportion of iso-amyl alcohol was 0.2%.The optimal culture conditions were as follows:slope growth time 8 d,seed age 36 h,inoculation amount 10%,fermentation days 7 d,culture temperature 28℃,and initial pH of fermentation medium 7.0.The fermentation titer of strain J-7-43 reached 924.9 mg/L.Then,the the production of josamycin in 5 L fermenter were investigated.The titer reached 1253.6 mg/L by increasing the initial rapeseed oil to 10%and maintaining the proportion of oil to 6%by feeding rapeseed oil during the fermentation stage.Furthermore,0.2%iso-amyl alcohol was added in the initial fermentation medium,and 0.1%isoamyl alcohol was fed every day from the 4th day,improving the titer of josamycin to 1586.3 mg/L.This strategy significantly reduced the ratio of 4"-O-butyryl-josamycin in the fermentation broth,and reduced the difficulty of downstream separation and purification.In addition,this study also sequenced the whole genome of J-2-40,and the biosynthetic gene cluster and biosynthetic pathway of josamycin ws deduced by bioinformatics analysis.Then,the effect of two post-modified genes orf 9 and orf 29 and acetyl-CoA carboxylase encoding gene accA2BE on the yield of josamycin was investigated,which made a preliminary exploration for the genetic modification of josamycin strains.更多还原