纳他霉素产生菌基因组重排选育高产菌株

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1、摘要纳他霉素(Natamycin)是一种多烯大环内酯类抗生素，能够有效抑制霉菌和酵母菌的生物活性，是有广阔应用前景的生物防腐剂和抗真菌药物。本论文以纳他霉素产生菌褐黄孢链霉菌SG．1和SG．2为出发菌株，应用基因组重排技术选育纳他霉素高产菌株，并对基因组重排选育方法的优越性进行检验。主要内容有以下几个方面：首先，建立了褐黄孢链霉菌的原生质体制备再生与融合的较优条件，采用双亲灭活的方法，进行两轮多亲本原生质体融合，通过琼脂块初筛和摇瓶复筛，最终得到1株高产重排菌株F2．2，其纳他霉素产量达到2．769／L

2、，比亲本菌株PS．3提高了64．3％，比原始出发菌株SG．2提高了97．1％。其次，分别考察了简单的双亲本原生质体融合、传统的原生质体制备与再生和基因组重排这三种选育方法对纳他霉素产量提高的影响，结果表明，两轮基因组重排后，获得的重排菌株F2．2的产量比亲本菌株PS．3的产量提高了64．3％，而两轮原生质体融合后，获得的融合菌株R2．1的产量比亲本菌株PS．3的产量只提高了30．9％，而反复的原生质体制备和再生不能诱发菌株的正突变。实验证明基因组重排技术是一种高效的菌种选育方法。关键词：褐黄孢链霉菌；纳

3、他霉素；原生质体融合：基因组重排ABSTRACTNatamycinisonekindofmacrolideantibiotics、析tIl26-memberedringpolyene8tnlc眦，whichhasabroadapplicationprospectasthebio-antisepticandantifungalagentforitsbiologicalactivitytorestrainthegrowthofmoldandyeast．Inthepaper,Streptomycesgilvo

4、sporeusSG一1andSG一2wereusedasthestartingstrains．ThengenomeshufflingWasusedtobreednatamycinhighproductionstrain，anditsefficiencywasverified．111emainresearchcontentsandresultswereasfollows．Firstly,nleconditionsforpreparation,regenerationandfusionoftheprotop

5、lastwereoptimized．Aftertwocyclesofrecursiveprotoplastfusion，theshuffledstrainF2-2whichwereselectedbyagarmethodandflakefermentationwasobtained,whosenatamycinproductionreached2．769／L，whichwasincreasedby64．3％and97．1％incomparison谢tllthatoftheparentalstrainPS

6、-3andoriginalstrainSG一2．．Secondly,threemethods，whichincludedsimpletwo-parentprotoplastfusion,traditionalprotoplastpreparationandregenerationandgenomeshuffling，wereusedtoimprovetheproductivityofnatamycinrespectively．11圮resultsshowed,aftertwocyclesofrecurs

7、iveprotoplastfusion,natamyeinproductionoftheshuffledstrainF2—2increasedby64．3％incomparisonwimthatoftheparentalstrainPS-3．Aftertwocyclesofsimpletwo-parentprotoplastfusion,natamycinproductionofthefusedstrainR2-lincreasedby30．9％incomparisonwitllthatofthepar

8、entalstrainPS-3．Protoplastpreparationandregenerationcouldnotleadtopositivemutation．Itprovedthatgenomeshufflingwasahigll●e舵ctivebreedingway．Keywords：&reptomycesgilvosporeus，Natamycin,Protoplastfusion,Genomeshuffling●目录1前言⋯⋯