代谢工程 英文原版PDF Chapter 9 - Methods for the Experimental De

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1、CHAPTER9MethodsfortheExperimentalDeterminationofMetabolicFluxesbyIsotopeLabelingInthepreviouschapterwepresentedthegeneralmethodologyforthedeterminationofintracellularmetabolicfluxesfromthemeasurementofextracellularmetaboliteconcentrations.Severalreasonswereprovidedtojustifythisexercise.Itwasnotedth

2、atintracellularfluxesareveryvaluableinestablishingthephysiologicalstateofabiologicalsystem.Aswewillshowinsubsequentchapters,metabolicfluxesplayanotherimportantrole,namely,theyprovideacomprehensivepictureofthecontrolarchitectureofmetabolicnetworks.Wewillshowthatmetaboliccontrolcoefficients,whichhave

3、beenproposedasmeasuresofthedegreeofcontrolexercisedinmetabolicnetworks,canbedetermineddirectlyfromthemagnitudeofmetabolicfluxesandfluxchangesinresponsetotheintroductionofmetabolicperturbations.Inthisregard,methodsfortheaccuratedeterminationofintracellularfluxesbecomeextremelyimportantastheycanleadd

4、irectlytotheelucidationofthecontroloffluxinmetabolicnetworks.353354MetabolicEngineeringTheprimaryinputsrequiredforthedeterminationofintracellularfluxes,asdiscussedinthepreviouschapter,arefluxesofextracellularmetabolites,namely,ratesofsubstrateuptakeandratesofproductsecretion.Theseratescanbestbeobta

5、inedfromsteadystatechemostatexperiments,inwhichconcentrationsofallmetabolitesaremeasuredatsteadystate.Thecorre-spondingratesofuptake/secretioncanthenbecalculatedbymultiplyingtheseconcentrationsbythechemostatdilutionrate.Becausechemostatexperimentsareverylonginduration,oftenbatchexperimentsareemploy

6、edforthedeterminationoffluxes.Batchcultivationofmicroorganismscangeneratelargeamountsofdatainarelativelyshortperiodoftime;however,thedeterminationofextracellularmetaboliteratesissignificantlymoreinvolvedasitrequirescalculationofthederivativeofthecorrespondingconcentrationswithrespecttotime,anoperat

7、ionthatusuallyintroduceslargeerrors.Furthermore,thelackofaphysiologicalsteadystateduringabatchcultureexperimentcancomplicatetheinterpretationofbatchculturedata.Whetherachemostatorabatchcultureexperimentisus