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鼠李糖脂减毒高产菌株的构建和盐霉素基因簇salBⅢ基因功能研究的开题报告.docx

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鼠李糖脂减毒高产菌株的构建和盐霉素基因簇salBⅢ基因功能研究的开题报告 Title:Constructionofareduced-toxicityandhigh-producingstrainofStreptomycessuaveolensforshuliangtangzhibiosynthesisandfunctionalstudyofthesalBIIIgeneclusterencodingthesalinomycinbiosynthesisgenes Introduction: ShuliangtangzhiisapolyetherionophoreantibioticproducedbythegenusStreptomycesandiswellknownforitsstrongactivityagainstGram-positivebacteria,fungi,andcancerstemcells.However,shuliangtangzhialsohasahighleveloftoxicity,limitingitsuseinclinicalmedicine.Inthisstudy,weaimtoconstructareduced-toxicityandhigh-producingstrainofStreptomycessuaveolensforshuliangtangzhibiosynthesisandperformfunctionalstudiesofthesalBIIIgeneclusterencodingthesalinomycinbiosynthesisgenes. Objectives: Theobjectivesofthisstudyareasfollows: -Toconstructareduced-toxicityandhigh-producingstrainofS.suaveolensbyintroducingmutationsintheshuilangtangzhibiosynthesisgenes -TocloneandsequencethesalBIIIgeneclusterfromS.suaveolens -ToperformfunctionalstudiesofthesalBIIIgeneclusterbygenedeletion,complementation,andoverexpressiontoidentifythefunctionofeachgeneinthecluster -Toevaluatetheantibacterialandantitumoractivitiesoftheshuliangtangzhiproducedbytheconstructedstrainandcompareittothewild-typestrain Methodology: 1.Constructionofreduced-toxicityandhigh-producingstrain: -Mutagenesisofshuliangtangzhibiosynthesisgenesbysite-directedmutagenesisorCRISPR-Cas9systemtoreducetoxicityandincreaseproductivity -SelectionandcharacterizationofthemutantswithreducedtoxicityandhighproductionthroughHPLC-MSanalysis 2.CloningandsequencingofthesalBIIIgenecluster: -DNAextractionandpurificationofthegenomicDNAofS.suaveolens -Amplification,cloning,andsequencingofthesalBIIIgeneclusterusingPCR 3.FunctionalstudiesofthesalBIIIgenecluster: -GenedeletionbyhomologousrecombinationusingtheRed/ETrecombinationsystem -Complementationbyintroducingacompletecopyofthedeletedgenebackintothemutantstrain -Overexpressionbyexpressingthegeneunderthecontrolofastrongpromoter 4.Evaluationofantibacterialandantitumoractivit