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江南大學:ARTP聯合高通量篩選來提高阿維菌素生產能力

作者:清華研究院生物育種中心 發布時間:2018/01/10 瀏覽量:2128

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  阿維菌素是一種具有廣譜活性、低毒性、高效的抗生素。作為一種生物殺蟲劑,阿維菌素在許多對農作物有害的昆蟲十分有效,不僅是用于家畜的抗寄生蟲藥,而且對人類寄生蟲病也有影響。由于這些優點,阿維菌素目前廣泛應用于動物保健、農業和人類感染。


  然而,由于相關的篩選工作量和時間限制,在工業應用領域很難使用隨機誘變和分子工程學來改造。因此,需要建立一些高通量篩選(HTS)方法來克服這些困難并有效地獲得正向突變菌株。

 

  江南大學周景文教授團隊利用ARTP技術對阿維鏈霉菌進行突變處理,通過反復實驗,確定了熒光染色最佳時間應為20分鐘,采用PIFDA作為熒光探針,對死孢子和活孢子進行鑒別。通過熒光標記后使用FACS進行篩選,從5760個突變體中篩選出19個高產菌株進行初篩,復篩后成功獲得阿維菌素高產突變體G9,(相比原始菌株,用搖瓶培養產量提高18.9%,用發酵罐培養產量提高20.6%)。

 

  此研究方法也可推廣到其他放線菌的改造篩選,通過FACS能進行高速篩選,后續利用多孔板培養,能縮短篩選時間2-3天。并且因為是通過誘變改良的菌株,沒有生物安全性的問題,很容易被應用到工業生產中

  

  上述研究成果得到了國家基礎研究發展計劃973計劃,項目標號2013 cb733602)中國國家自然科學基金項目編號21390204),江蘇省重點研究開發項目項目編號BE2016689中央高校基本科研專項資金項目編號JUSRP51701A江蘇省六大人才高峰項目(項目編號2015-JY-005等相關項目資助。

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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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