《Expression and Characterization of ArgR, An Arginine Regulatory Protein in Corynebacterium crenatum＊.》.pdf

436 BiomedEnvironSci，2014；27(6)：436-443 OriginalArticle ExpressionandCharacterizationofArgR，AnArginine RegulatoryProteininCorynebacterium crenatum CHEN XueLan，ZHANGBin，TANG Li，JIAOHaiTao，XU HengYi，XU Feng， XU Hong，WEIHua，andXIONG YongHua2， 1．KeyLaboratoryofFunctionalSmallOrganicMolecule,MinistryofEducation,Jidn iNormalUniversity, Nanchang330022，~angxi,China,2．StateKeyLaboratoryofFoodScienceandTechnology,NanchangUniversity Nanchang330047,Jiangxi,China,3．OceanNanoTech,LLC,Springdale．AR72764,USA Abstract Objective Corynebacterium crenatum MT_amutantfromC crenatumAS1．542withalethalargR gene．exhibitshigharginineproduction．To cOnfirm theeffectofArgR onargininebiosynthesisinC crenatum，anintactargRgenefromwild—typeAS1．542wasintroducedintoCcrenatum MTJresultingin C crenatum MT．sp，andthechangesoftranscriptionallevelsoftheargininebiosyntheticgenesand arginineproductionwerecomparedbetweenthemutantstrainandtherecombinantstrain． Methods Quantitativereal—timepolymerasechainreactionwasemployedtoanalyzethechangesof the related genes atthe transcriptionallevel，electrOphOreticmobility shift assayswere used to determine ArgR binding with the argCJeDF,argGH,and carAB promoter regions，and arginine productionwasdeterminedwithanautomatedaminoacidanalyzer． Results Arginineproductionassaysshoweda69．9％reductioninargininefrom9．01~0．22mg／mLinc crenatumMTt02．71~0．13mg／mL(PO．05)inCcrenatum MT．sp．TheargCargB，argD，argF,argJ， argG，andcarAgenesweredown-regulatedsignificantlyinC crenatum MT．spcomparedwiththosein itsparentalC crenatum MTstrain．TheeIectrOphOreticmobilityshiftassaysshowedthatthepromoter regionsweredirectlyboundtotheArgRprotein． Conclusion Thearginine biosyntheticgenesinC crenatum areclearlycontrolled bythe negative regulatorArgR，and intactArgR in C crenatum MT resultsin a significantdescrease i