代谢工程的课件资料

代谢工程的课件资料第1页/共30页RecombinantproteinsbymicroorganismsTransformationRestrictionsitesCloningvectorLigationRecombinedplasmidRestrictioncleavageRestrictioncleavageGeneofinterestTranscriptionTranslationmRNAProteinHostcell第2页/共30页Recombinant

proteinsbymicroorganismsYear Products Disease Company1982 Humulin Type1diabetes Genentech,Inc. (syntheticinsulin)

1985 Protropin Growthhormone Genentech,Inc. Deficiency

Someearlyproducts第3页/共30页WhatisMetabolicEngineering?

Metabolicengineeringisreferredtoasthedirectedimprovementofcellular

propertiesthroughthemodificationofspecificbiochemicalreactionsortheintroductionofnewones,withtheuseofrecombinantDNAtechnology

第4页/共30页ThreemajorprojectsPathwayandco-factorengineeringQuantitativesystemsbiotechnology (genomics,transcriptomics,proteomicsandmetabolomics)Plantmetabolicengineering第5页/共30页GeneTargetsIdentifiedInsert/KnockoutGenes/pathwaysPotentialstrainsShakeFlask/BioreactorExperimentsAnalysisModelingMolecularBiologyTechniquesPCR,REdigest,ligation,transformation,knockout,etc.MetabolicEngineering/systemsbiotechnologyCycleMetabolicevolutionmetabolomicsproteomicstranscriptomics第6页/共30页Pathwaydesignandmanipulation第7页/共30页Succinicacid第8页/共30页TopValueAddedChemicalsfromBiomass(2004DOEreport)

第9页/共30页TopValueAddedChemicalsfromBiomass(2004DOEreport)

第10页/共30页SimplifiedcentralanaerobicpathwayinE.coliNADHlimitationMaxtheoreticalYield1mole/moleSuccinate

2NADH

2NAD+

Lactate2NAD+

2NADH

FormatePyruvate

Acetyl-CoAAcetateFructose1,6-diPGlucoseGlucose-6-PBiomassPEPPyruvateADPATPAcetyl-PADPATPPEPADPATPGlycerate-1,3-diPADPATPOAA

CoANADHNAD+

EthanolNADH

CO2

H2NAD+PepcLdhPta

AckCO2第11页/共30页

E.coliWildTypesuccinateProblems:lowyield;mixedacidfermentation第12页/共30页

ResultingpathwaydesignSimplifiedcentralanaerobicpathwayinE.coliLactateAcetyl-PAcetateADPATPAckSuccinate

2NADH

2NAD+

2NAD+

2NADH

FormatePyruvate

Acetyl-CoA

Fructose1,6-diP

GlucoseGlucose-6-PBiomassPEPPyruvate

ADPATPPEPADP

ATP

Glycerate-1,3-diPADPATPOAA

CoAEthanol

NADH

CO2

NAD+PepcLdhPta

H2CO2NADHNAD+

NADHlimitationMaxtheoreticalYield1mole/moleFermentativepathwayGlyoxylatepathway(noNADHrequirements)aceAIsocitrateOAAMalate

SuccinateNAD+

NADH

glyoxylateAcetyl-CoA

aceB

Fumarate

NADH

NAD+

aceA

CitratePycCO2Fructose1,6-diP

NADHPyruvate

Pfl

ADP

ATP

Glycerate1,3-diP

Glucose

Glucose-6P

PEP

Pyruvate

ADP

ATP

NAD+PykPEP

Acetyl-CoA

H2CO2FormateADP

ATP

CO2Experimentalyield=1.6mol/mol第13页/共30页

Resultingpathwaydesign

FermentativepathwayGlyoxylatepathway(noNADHrequirements)PYCCO2aceAIsocitrateOAA

Malate

Succinate

NAD+

NADH

-

glyoxylateAcetylCoA

aceB

Fumarate

NADH

NAD+

aceA

CitrateFructose1,6-diP

NADHPyruvate

PFL

ADP

ATP

Dihydroxyacetone-PGlyceraldehyde-3P

Glycerate1,3-diP

Glucose

Glucose-6P

PEP

Pyruvate

ADP

ATP

Pi+NAD+

PykPEP

Acetyl-CoA

H2CO

2

Formate

ADP

ATP

CO2第14页/共30页SBS550MG(pHL413)Succinicacidproducedfrom20g/L(~110moles)ofglucoseE.coliWildTypesuccinatesuccinate第15页/共30页ThreeroutesengineeredforsuccinateproductionStrainSBS552Genesinactivated:sdhABackA-ptapoxBiclRptsGldhAadhE(aerobic)(anaerobic)(anaerobic&aerobic)PyruvateAcetyl-CoASuccinateG6PPEP

Pyruvate

PEPG3PCO2

GlucoseOAACitrateIsocitrateMalateGlyoxylateAcetyl-CoAaceAaceBCO2

ptsGicd2-ketoglutarateSuccinyl-CoACO2CO2FumarateSuccinateSuccinateCO2

第16页/共30页Cofactormanipulations第17页/共30页Cofactormanipulations•NAD+/NADH;NADP+/NADPH•CoenzymeA(CoA)第18页/共30页Examplesesterschiralcompoundsterpenoids–lycopenepolyketidesCancer(adriamycin)Infectiondisease(tetracyclines,erythromycin)Cardiovascular(mevacor,lovastatin)Immunosuppression(rapamycin,tacrolimus)第19页/共30页ImportanceofcofactormanipulationEnzymesCofactors+ProductsSubstrate第20页/共30页LycopeneproductionislimitedbyNADPHavailability

136.0%(2.4)136.7%(2.4)WTmutantExample:lycopeneproduction第21页/共30页Plantmetabolicengineering第22页/共30页MetabolicEngineeringoftheTerpenoidandIndolePathwaysinCatharanthusroseushairyrootsCatharanthusroseus(MadagascarPeriwinkle)ProduceawiderangeofsecondarymetabolitesAjmalicineandSerpentine–hypertensionVinblastineandVincristine–anticancerdrugsusedtotreatlymphomasandleukemiavinblastinevincristine第23页/共30页ChorismateAnthranilateTryptophanTryptaminePyruvate+G3P1-Deoxy-D-Xylulose-5-Phosphate2-C-Methyl-D-erythitol-4-phosphateIPPDMAPPGPPGeraniol10-HydroxygeraniolLoganinSecologaninStrictosidineAjmalicineSerpentineTabersonineLochnericineHörhammericineVindolineCatharanthineVinblastineVincristineTerpenoidIndoleAlkaloidPathwayIndole

PathwayTerpenoidPathwayMevalonateASDXSSTRG10HTDC第24页/共30页CloneGenerationPlasmidConstructioninE.coliAgrobacteriumRiSterileGrownPlants(5weeks)

Infection(6weeks)SelectionMedia(6weeks)AdapttoLiquidMedia(24weeks)Transgene第25页/共30页DiscoveryofnewcompoundsIsolatedandverifiedthestructuresof11newphenoliccompoundsStructureverysimilartoexistingagentsthatex