Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains.docVIP

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Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains.doc

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Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains

ApplicationofaGeneticallyEncodedBiosensorforLive CellImagingofL-ValineProductioninPyruvate DehydrogenaseComplex-DeficientCorynebacterium glutamicumStrains NurijeMustafi1.,AlexanderGru¨nberger1.,ReginaMahr1,StefanHelfrich1,KatharinaNo¨h1, BastianBlombach2,DietrichKohlheyer1,JuliaFrunzke1* 1IBG-1:Biotechnology,ForschungszentrumJu¨lich,Ju¨lich,Germany,2InstituteofBiochemicalEngineering,UniversityofStuttgart,Stuttgart,Germany Abstract The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently,theanalysisofmicrobialmetaboliteproductionisstilldominatedbybulktechniques,whichmayobscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum DaceE . Online monitoringofthesensoroutput(eYFPfluorescence)duringbatchcultivationprovedthesensor’ssuitabilityforvisualizing differentproductionlevels.Inthefollowing,weconductedlivecellimagingstudiesonC.glutamicumsensorstrainsusing microfluidicchipdevices.Asexpected,thesensoroutputwashigherinmicrocoloniesofhigh-yieldproducersincomparison tothebasicstrainC.glutamicumDaceE.MicrofluidiccultivationinminimalmediumrevealedatypicalGaussiandistribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changedtheobservedphenotypicpatternofallstrains,resultinginaphenotypicsplitofthepopulation.Whereassome cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggestingalossofmetabolicactivity.Thesestudiesdemonstratethatgeneticallyencodedbiosensorsareavaluabletool formoni