产甲基丙二酰辅酶A假单胞菌的构建和雷帕霉素生物合成基因簇的定位的中期报告Abstract:TheconstructionofaPseudomonasputidastraincapableofoverproducingmethylmalonyl-CoAforthebiosynthesisofrapamycinwasaimedat.Methylmalonyl-CoAisaprecursorforthebiosynthesisofbothrapamycinandprodigininesandisthereforeakeytargetforincreasedproduction.Toachievethisgoal,weutilizedtheCRISPR-Cas9systemtoknockoutthepropionatepathwayinP.putidaandreplaceditwithaheterologouspathwayfortheproductionofmethylmalonyl-CoA.Wehavesuccessfullyconstructedarecombinantstrainwhichcanaccumulatemoremethylmalonyl-CoAthanthewild-typestrain.Introduction:RapamycinisamacrocyclicpolyketideproducedbyStreptomyceshygroscopicusandhasimmunosuppressiveandanticancerproperties.Duetothehighdemandforrapamycininmedicalapplications,thereisaneedforimprovedproductionmethods.Onewaytoachievethisisbyincreasingtheprecursorsupplyforthebiosynthesisofrapamycin.Methylmalonyl-CoAisakeyprecursorforthebiosynthesisofbothrapamycinandprodiginines,whichareredpigmentssynthesizedbyvariousbacteriaandarepotentialdrugtargets.Therefore,increasingthesupplyofthisprecursoriscriticalfortheincreasedproductionofbothrapamycinandprodiginines.Results:WeconstructedarecombinantPseudomonasputidastrainwhichcanproducemethylmalonyl-CoAbyknockingoutthepropionatepathwayandreplacingitwithaheterologouspathwayfortheproductionofmethylmalonyl-CoA.TherecombinantstrainwasconfirmedbyPCRandsequencinganalysis.Theproductionofmethylmalonyl-CoAwasmeasuredbyHPLCandLC-MS.Therecombinantstrainwasfoundtoaccumulatehigherlevelsofmethylmalonyl-CoAcomparedtothewildtypestrain.Furtheranalysisisongoingtooptimizetheproductionofmethylmalonyl-CoAanditsuseforthebiosynthesisofrapamycinandprodiginines.Conclusions:TheconstructionoftheP.putidastraincapableofoverproducingmethylmalonyl-CoAforthebiosynthesisofrapamycinispromisingforthepotentialcommercialproductionofrapamycin.Theoptimizationofthisstrainforthebiosynthesisofprodigininesisalsoimportantforfutureresearch.Thisstudyprovidesafoundationfortheincreasedproductionofmethylmalonyl-Co