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江南大学:ARTP联合高通量筛选来提高阿维菌素生产能力

作者:清华研究院生物育种中心 发布时间:2018/01/10 浏览量:346

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  阿维菌素是一种具有广谱活性、低毒性、高效的抗生素。作为一种生物杀虫剂,阿维菌素在许多对农作物有害的昆虫十分有效,不仅是用于家畜的抗寄生虫药,而且对人类寄生虫病也有影响。由于这些优点,阿维菌素目前广泛应用于动物保健、农业和人类感染。


  然而,由于相关的筛选工作量和时间限制,在工业应用领域很难使用随机诱变和分子工程学来改造。因此,需要建立一些高通量筛选(HTS)方法来克服这些困难并有效地获得正向突变菌株。

 

  江南大学周景文教授团队利用ARTP技术对阿维链霉菌进行突变处理,通过反复实验,确定了荧光染色最佳时间应为20分钟,采用PIFDA作为荧光探针,对死孢子和活孢子进行鉴别。通过荧光标记后使用FACS进行筛选,从5760个突变体中筛选出19个高产菌株进行初筛,复筛后成功获得阿维菌素高产突变体G9,(相比原始菌株,用摇瓶培养产量提高18.9%,用发酵罐培养产量提高20.6%)。

 

  此研究方法也可推广到其他放线菌的改造筛选,通过FACS能进行高速筛选,后续利用多孔板培养,能缩短筛选时间2-3天。并且因为是通过诱变改良的菌株,没有生物安全性的问题,很容易被应用到工业生产中

  

  上述研究成果得到了国家基础研究发展计划973计划,项目标号2013 cb733602)中国国家自然科学基金项目编号21390204),江苏省重点研究开发项目项目编号BE2016689中央高校基本科研专项资金项目编号JUSRP51701A江苏省六大人才高峰项目(项目编号2015-JY-005等相关项目资助。

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Fig. 1 Determination of a preliminary screening method.a Absorptionscanning of avermectin. Wide spectrum scans to determine theappropriate concentrations of avermectin standard and culture medium wereperformed. There was an absorption peak at OD245 nm due to the avermectin standard (solid line). Interference ofthe culture medium (dotted line) at OD245 could be nearly neglected. b Relationship between OD245 and avermectinconcentration. The avermectin was diluted to an appropriate concentration withanhydrous methanol. There was a good linear relationship between avermectinconcentration and  D245

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Fig. 3 Control of PIand FDA.aThe R1 gatemarked the main position of target spores in the scatter plot. b The backgroundof spores was set as the negative control. c R2 was set as the PI singlepositive area. PI was used to stain absolutely dead spores; the fluorescencewas mainly in the R2 section. d R5 was set as the FDA single positive area. FDAwas used to stain live spores; the fluorescence was mainly in the R5 section

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Fig. 6 ARTP mutation primary screening. Atotal of 19 strains were selected for primary screening from 5760 mutants. CKrepresents control strain. The significant difference analysis of other strainswas done compared to control strain. Each point represents the mean of threebiological replicates and error bars indicate the standard derivation.Differences are considered as significant when P value < 0.05(*) or <0.01(**)

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Fig. 7 Second screening in the shakeflasks. Production of avermectin B1a by the optimum strain G9 was improved by18.9% compared to the control strain (CK). The significant difference analysisof other strains was done compared to control strain. Each point represents themean of three biological replicates and error bars indicate the standard derivation.Differences are considered as significant when P value < 0.05(*) or <0.01(**)

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Fig. 8 Production of avermectin in the 15-Lfermenter. Comparison of the original strain and the G9 mutant in a 15-Lfermenter. The original strain and the G9 mutant were cultivated for 281 h in a15-L fermenter at 28 ℃. Avermectin production was 20.6% higher in the mutant than in theoriginal strain. Glucose (G9 mutant, gray squares; original strain, blacksquares); pH (G9 mutant, gray triangles; original strain, black triangles);avermectin (G9 mutant, gray circles; original strain, black circles)

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