Divisions of Bioengineering Departments of Chemical Engineering Rice University Houston, Texas

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Metabolic Engineering and Systems Biotechnology Ka-Yiu San Departments of Bioengineering Departments of Chemical Engineering Rice University Houston, Texas

Recombined plasmid Restriction cleavage mRNA Gene of intrigue Translation Restriction destinations Ligation Transcription Protein Restriction cleavage Transformation Cloning vector Host cell Cloning for rProtein generation

Recombinant 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

Examples of a couple biopharmaceutical items in 1994 Source: Biotechnology Industry Organization, Pharmaceutical Research and Manufacturers of America, organization comes about, investigator reports

What 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

Protein/catalyst Gene mRNA interpretation Modern science – focal authoritative opinion interpretation

Protein/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

NADH (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

Current Projects

Cofactor designing

Motivations 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

Enzymes + Cofactors Substrate Products Importance of cofactor control

Cofactor building NAD +/NADH CoA/acetyl-CoA

NADH (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

Coenzyme 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

Acetyl-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

Lactic corrosive Polylactic corrosive (PLA) LDH Pyruvate Lactate NAD + NADH Example: Lactic corrosive arrangement

Biopolymer 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)

Polyketide 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

Polyketide creation Precursor supply - case Ref: Precursor Supply for Polyketide Biosynthesis: The Role of Crotonyl-CoA Reductase, Metabolic Engineering 3, 40-48 (2001)

Approach 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

Manipulation of NADH accessibility

Glucose NAD + Succinate 2NADH Pyruvate Lactate NADH Acetyl-CoA Formate 2NADH Acetate Ethanol 2NAD + NAD + NADH 2NAD + Fermentation Pathway of E. coli

Pyruvate 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

Construction of pSBF2 Overexpressing FDH pFDH1 PCR fdh pSBF2 XbaI pSBF2 fdh EcoRI/XbaI pUC18 pUCFDH XbaI pDHK30

Strain 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

XbaI 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)

Anaerobic 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

NADH 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

NADH Availability 5.0 4.0 mol NADH/mol glucose 3.0 2.0 1.0 0.0 GJT(pDHK29) GJT(pSBF2)

Summary 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

Quantitative frameworks biotechnology

Projects 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

Motivations 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

Motivations 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?

Genome Database Pathway Database FBA Metabolic Pattern Metabolic Network A priori Knowledge Traditional flux adjust investigation (FBA)

Metabolic Network (From http://www.genome.ad.jp/kegg/pathway/delineate)

Metabolic Pattern (Illustration) 1.0 0.8 0.2 0.8: Metabolic rates (From http://www.genome.ad.jp/kegg/pathway/outline)

genotype 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

? 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

Model System Oxygen and redox detecting/direction framework Sugar use administrative system

Simplified 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

e - 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)

Some 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