Metabolic Engineering and Systems Biotechnology Ka-Yiu San Departments of Bioengineering Departments of Chemical Engineering Rice University Houston, Texas
Slide 3Recombined plasmid Restriction cleavage mRNA Gene of intrigue Translation Restriction destinations Ligation Transcription Protein Restriction cleavage Transformation Cloning vector Host cell Cloning for rProtein generation
Slide 4Recombinant proteins by microorganisms Some early items Year Products Disease Company 1982 Humulin Type 1 diabetes Genetech, Inc. (manufactured insulin) 1985 Protropin Growth hormone Genetech, Inc. Inadequacy
Slide 5Examples of a couple biopharmaceutical items in 1994 Source: Biotechnology Industry Organization, Pharmaceutical Research and Manufacturers of America, organization comes about, investigator reports
Slide 6What is metabolic building? Metabolic building is alluded to as the coordinated change of cell properties through the alteration of particular biochemical responses or the presentation of new ones, with the utilization of recombinant DNA innovation
Slide 7Protein/catalyst Gene mRNA interpretation Modern science – focal authoritative opinion interpretation
Slide 8Protein/chemical Gene mRNA translation interpretation Current metabolic designing methodologies Amplification of chemical levels Use catalysts with various properties Addition of new enzymatic pathway Deletion of existing enzymatic pathway Genetic control
Slide 9NADH (Reduced) NAD + (Oxidized) Current undertakings Cofactor building of Escherichia coli Manipulation of NADH accessibility Manipulation of CoA/acetyl-CoA Plant metabolic building 3. Quantitative frameworks biotechnology A. Rational pathway outline and improvement Metabolic flux examination in view of element genomic data Design and demonstrating of counterfeit hereditary systems Metabolite profiling Genetic systems – structures and physiology
Slide 10Current Projects
Slide 11Cofactor designing
Slide 12Motivations and speculation Motivations Existing metabolic designing procedures incorporate pathway erasure pathway option pathway adjustment: enhancement, tweak or utilization of isozymes (or catalyst from coordinated advancement consider) with various enzymatic properties Cofactors assume a crucial part in an expansive number of biochemical responses Hypothesis Cofactor control can be utilized as an extra apparatus to accomplish wanted metabolic designing objectives
Slide 13Enzymes + Cofactors Substrate Products Importance of cofactor control
Slide 14Cofactor building NAD +/NADH CoA/acetyl-CoA
Slide 15NADH (Reduced) NAD + (Oxidized) NADH/NAD + Cofactor Pair Important in digestion system Cofactor in > 300 red-bull responses Regulates qualities and compounds Donor or acceptor of diminishing reciprocals Reversible change Recycle of cofactors fundamental for cell development
Slide 16Coenzyme A (CoA) Essential intermediates in numerous biosynthetic and vitality yielding metabolic pathways CoA is a transporter of acyl gathering Important part in enzymatic generation of mechanically valuable mixes like esters, biopolymers, polyketides and so forth
Slide 17Acetyl-CoA Entry indicate Energy yielding TCA cycle Important segment in unsaturated fat digestion system Precursor of malonyl-CoA, acetoacetyl-CoA Allosteric activator of specific chemicals
Slide 18Lactic corrosive Polylactic corrosive (PLA) LDH Pyruvate Lactate NAD + NADH Example: Lactic corrosive arrangement
Slide 19Biopolymer creation Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/PHV piece copolymer) Glycerol Propionate Acetyl-CoA Propionyl-CoA Acetyl - CoA 3-Ketothiolase (PhaA) HSCoA Acetoacetyl-CoA 3-Ketovaleryl-CoA NADPH Acetoacetyl-CoA Reductase (PhaB) NADP + 3-Hydroxybutyryl-CoA 3-Hydroxyvalery-CoA PHA Synthase (PhaC) HSCoA P(HB-co-HV)
Slide 20Polyketide creation Complex common items > 10,000 polyketides recognized Broad scope of helpful applications Cancer (adriamycin) Infection sickness (antibiotic medications, erythromycin) Cardiovascular (mevacor, lovastatin) Immunosuppression (rapamycin, tacrolimus) 6-deoxyerythronolide B
Slide 21Polyketide creation Precursor supply - case Ref: Precursor Supply for Polyketide Biosynthesis: The Role of Crotonyl-CoA Reductase, Metabolic Engineering 3, 40-48 (2001)
Slide 22Approach Systematic control of cofactor levels by hereditary designing means Model frameworks Simple model frameworks, for example, biosynthesis of succinate and ester, to represent the idea Results expanded NADH accessibility to the phone expanded levels of CoA and acetyl CoA altogether change metabolite redistribution
Slide 23Manipulation of NADH accessibility
Slide 24Glucose NAD + Succinate 2NADH Pyruvate Lactate NADH Acetyl-CoA Formate 2NADH Acetate Ethanol 2NAD + NAD + NADH 2NAD + Fermentation Pathway of E. coli
Slide 25Pyruvate NAD + NADH PFL Formate CO 2 FDH1 Acetyl-CoA FDHF CO 2 unique NAD free pathway (FDHF: formate dehydrogenase, NAD autonomous) Newly included NAD+ subordinate pathway (FDH1: NAD+ subordinate formate dehydrogenase FDH1 encoded by fdh1 from Candida boidinii) H 2 NADH Regeneration
Slide 26Construction of pSBF2 Overexpressing FDH pFDH1 PCR fdh pSBF2 XbaI pSBF2 fdh EcoRI/XbaI pUC18 pUCFDH XbaI pDHK30
Slide 27Strain FDH action (units/mg protein) GJT001(pSBF2) 0.42 BS1(pSBF2) 0.28 GJT001(pDHK29) Not distinguished BS1(pDHK30) Not recognized Assay of FDH action
Slide 28XbaI lacZ' Km R lacZ fdh MCS pSBF2 pDHK29 Km R Ori Characterization of NADH-ward FDH NADH-subordinate FDH NADH-subordinate FDH PanK GJT (pDHK29) (Control strain) GJT (pSBF2) (New strain)
Slide 29Anaerobic Tubes : Experimental Method Strains : Escherichia coli (MC4100 subsidiary) GJT001 (pDHK29): wild sort (control plasmid) GJT001 (pSBF2): wild sort (new FDH plasmid) Media: LB + 1g/L NaHCO 3 100mg/L Kanamycin 20g/L Glucose Temperature: 37 ºC Agitation: 250 rpm Samples: 72 hrs after vaccination HPLC
Slide 30NADH 2NAD+ NAD+ 2NAD+ Effect of Increasing NADH Availability % of Increase/Decrease for GJT001 (pSBF2) with respect to GJT001 (pDHK29) Glucose Consumed NAD+ 3-overlap Succinate 55% 2NADH Lactate Pyruvate 91% NADH Formate Converted Acetyl-CoA 2NADH Acetate 8-crease 43% NADH NAD + CO 2 Formate Ethanol O.D.600nm: 59% Et/Ac: 27-overlay FDH1 FDHF 15-crease CO 2 H 2
Slide 31NADH Availability 5.0 4.0 mol NADH/mol glucose 3.0 2.0 1.0 0.0 GJT(pDHK29) GJT(pSBF2)
Slide 34Summary of results Effect of NADH recovery (overexpressing NAD + - subordinate FDH): Increases intracellular NADH accessibility Provide a more diminished environment Increase lessened item, (for example, ethanol and succinate) efficiency altogether
Slide 35Quantitative frameworks biotechnology
Slide 36Projects Metabolic flux investigation in view of element genomic data Rational pathway outline and advancement attainable and feasible new system plan Design and demonstrating of fake hereditary systems
Slide 37Motivations Observations Traditional reductionist approach Knowledge at the essential and basic level – however for the most part detached Information flood Genome database, quality expression database (utilitarian genomic), proteomic, metabolomics, metabolic pathway database Most of the current information base – static Genome database, metabolic pathway database
Slide 38Motivations and destinations: How would one be able to use the static genomic and metabolic databases (particularly when hereditary/administrative system structures are accessible) to portray and anticipate cell capacities, for example, metabolic examples?
Slide 39Genome Database Pathway Database FBA Metabolic Pattern Metabolic Network A priori Knowledge Traditional flux adjust investigation (FBA)
Slide 40Metabolic Network (From http://www.genome.ad.jp/kegg/pathway/delineate)
Slide 41Metabolic Pattern (Illustration) 1.0 0.8 0.2 0.8: Metabolic rates (From http://www.genome.ad.jp/kegg/pathway/outline)
Slide 42genotype phenotype ecological hereditary annoyances irritations (mutant strains) Cellular Responses Transcription Translation Metabolic Flux Analysis OR Metabolite Patterns Protein/chemical Gene mRNA Stimuli conventional metabolic building study
Slide 43? Genome Database Pathway Database Genetic Structure Expression Patterns Genetic Network A priori Knowledge FBA Metabolic Network Metabolic Patterns Gene Regulation Knowledge Gene Chip (Array) Data Proposed New Approach Environmental Conditions
Slide 44Model System Oxygen and redox detecting/direction framework Sugar use administrative system
Slide 45Simplified schematic of E. coli focal metabolic pathways Glucose PEP Pyruvate Lactate ldhA [1.1.1.28] NAD + ,CoA ppc [4.1.1.31] NADH, CO 2 pdh [1.2.4.1] CoA pfl [2.3.1.54] H 2 + CO 2 Formate CO 2 Acetyl-CoA Ethanol gltA [4.1.3.7] Acetate Citrate Oxaloacetate aspC [2.6.1.1] NADH acnB [4.2.1.3] mdh [1.1.1.37] NADH NAD + NAD+ Isocitrate Aspartate Malate aspA [4.3.1.1] fumB [4.2.1.2] fumA [4.2.1.2] icd [1.2.4.2] NADP + NADPH Fumarate sdhCDAB [1.3.99.1] CO 2 NADH frdABCD [1.3.1.6] 2-ketoglutarate NAD + sucAB [1.2.4.2] Succinate sucCD [6.2.1.5] NAD + NADH Succinyl-CoA CO 2 Simplified schematic of E. coli focal metabolic pathways
Slide 46e - transport Cytoplasmic film ArcB P FNR Redox, metabolites Aer Redox? Dos ArcA O 2 O 2 CheW,A,Y ArcA-P Transcription obscure Energy taxis Transcription Schematic demonstrating chose oxygen and redox detecting pathways in E . coli (received from Sawers, 1999)
Slide 47Some case of accessible pathway data FNR dynamic without oxygen; ArcA is enacted without oxygen Ref 1: "Reg of quality expression in fermentative and respiratory frameworks in Escherich
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