Chapter 5: Major Metabolic Pathways

1 1 Michigan Technological UniversityDavid R. Shonnard1 Chapter 5: Major Metabolic PathwaysDavid ShonnardDepartment of Chemical EngineeringMichigan Technological UniversityMichigan Technological UniversityDavid R. Shonnard2 Presentation Outline:lIntroduction to MetabolismlGlucose MetabolismGlycolysis, Kreb s Cycle, RespirationlBiosysthesislFermentation2 Michigan Technological UniversityDavid R. Shonnard3 Metabolismis the collection of enzyme-catalyzed reactions that convert substrates that are external to the cell into various internal Technological UniversityDavid R. Shonnard4 Genetic Engineeringallows for the alteration of metabolism by insertion or deletion of selected genes in a predetermined manner ( Metabolic Engineering).

2 An understanding of Metabolic Pathways in the organism of interest is of primary importance in bioprocess development. Introduction: Metabolism, Genetic Engineering and Bioprocessing3 Michigan Technological UniversityDavid R. Shonnard5 Characteristics of Metabolism1. Varies from organisms to organism2. Many common characteristics3. Affected by environmental conditions a) O2availability: Saccharomyces cerevisiaeAerobic growth on glucose more yeast cellsAnaerobic growth on glucose ethanol b) Control of metabolism is important in bioprocessesMichigan Technological UniversityDavid R. Shonnard6 Types of MetabolismCatabolismMetabolic reactions in the cell that degrade a substrate into smaller / simpler CO2 AnabolismMetabolic reactions that result in the synthesis of larger / more complex Technological UniversityDavid R.

3 Shonnard7 Figure : Classes of Reactions (Fig. ) CatabolismAnabolism Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 2002 Michigan Technological UniversityDavid R. Shonnard8 Bioenergetics The source of energy to fuel cellular metabolsim is reduced forms of carbon (sugars, hydrocarbons, etc.)The Sunis the ultimate source via the process of Photosynthesisin plantsCO2+ H2O + hv CH2O + O25 Michigan Technological UniversityDavid R. Shonnard9 ATP - Adenosine TriphosphateCatabolism of carbon-containing substrates generates high energy biomoleculesadenineribose3 high -energy phosphate bonds Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 2002 Michigan Technological UniversityDavid R.

4 Shonnard10 ATP - ReactionsStorage of energyRelease of energyATP + H2O ADP + Pi; Go= kcal/moleADP + H2O AMP + Pi; Go= kcal/moleAnalogs of ATPGTP = guanosine triphosphateUTP = uridine triphosphateCTP = cytidine triphosphate6 Michigan Technological UniversityDavid R. Shonnard11 ATP: Energy Currency of the Cell (Fig. ) Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 2002 Michigan Technological UniversityDavid R. Shonnard12 NAD+and NADP + (Fig. ) Nucleotide derivatives that accept H+and e-during oxidation / reduction reactions Transfer e-to O2during respiration Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 20027 Michigan Technological UniversityDavid R.

5 Shonnard13 Glucose Metabolism:CatabolicPathways of Primary Importance1. Glycolysis: from glucose to Krebsor tricarboxylic acid (TCA) cycle for conversion of pyruvate to Respirationor electron transport chainfor formation of ATP by transferring electrons from NADH to an electron acceptor (O2under aerobic conditions).Michigan Technological UniversityDavid R. Shonnard14 Glycolysis: Embden-Meyerhof-Parnas (EMP) pathway Principles of Biochemistry , Lehninger, Worth 8 Michigan Technological UniversityDavid R. Shonnard15 Glycolysis: in Eucaryotes Fermentation of Glucose Pyruvate no O2required Occurs in the CytoplasmGlucose + 2 ADP + 2 NAD++ 2 Pi 2 Pyruvate + 2 ATP + 2 (NADH + H+)2 (FADH + H+)4 ATP = 6 ATPIn Eucaryotes, Cytoplasm to MitochondriaMichigan Technological UniversityDavid R.

6 Shonnard16 Krebs or TCA Cycle In Mitochondria of eucaryotes provides e-(NADH) and ultimately energy (ATP) for biosynthesis provides intermediates for amino acid synthesis generates energy (GTP)9 Michigan Technological UniversityDavid R. Shonnard17 Krebs or TCA Cycle(Fig. ) Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 2002 Michigan Technological UniversityDavid R. Shonnard18 Krebs or TCA Cycle Pyruvate + 4 NAD++ FAD 3 CO2+ 4 NADH2+ FADH2 GDP + Pi GTPGTP + ADP GDP + ATPY ield of ATP1 + 4(3) + 2 = 15 ATP10 Michigan Technological UniversityDavid R. Shonnard19 Complete Oxidation of Glucose Glucose + 36 Pi+ 36 ADP + 6 O2 6 CO2+ 6 H2O + 36 ATP Go= (36)( kcal/mole) = 263 kcal/mole glucoseMichigan Technological UniversityDavid R.

7 Shonnard20 Energetics of Glucose Oxidation Direct Oxidation of GlucoseGlucose + 6 O2 6 CO2+ 6 H2O Go= 686 kcal/mole glucoseEnergy Efficiency of Glycolysis/TCA Cycle263/686(100) = 38% (standard conditions) 60% (nonstandard conditions)11 Michigan Technological UniversityDavid R. Shonnard21 ATP Yields Eucaryotes3 ATP, 2 ATP 36 ATPNADH FADH GlucoseProcaryotes2 ATP, 1 ATP 24 ATPNADH FADH GlucoseMichigan Technological UniversityDavid R. Shonnard22 Respiration In Mitochondria of eucaryotes in membrane-bound proteins in procaryotes e-transport from NADH or FADH to an electron acceptorAerobicO2 AnaerobicNO3-, SO42-, Fe3+, Cu2+, So, 12 Michigan Technological UniversityDavid R.

8 Shonnard23 Respiration(Fig. ) Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 2002 Oxidative PhosphorylationGoals of Respiration1. Regenerate NAD+2. Generate ATPM ichigan Technological UniversityDavid R. Shonnard24 Biosynthesis The EMP pathway and TCA cycle are used for catabolism (Glucose CO2+ NADH + ATP) primarily. energy production. The Hexose - Monophosphate pathway (HMP) is used for biosynthesis13 Michigan Technological UniversityDavid R. Shonnard25 HMP pathway (Fig. )** Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 2002 Michigan Technological UniversityDavid R. Shonnard26 Amino Acids by Various Pathways (Fig.)

9 Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 200214 Michigan Technological UniversityDavid R. Shonnard27 Fermentation:No TCA Cycle or RespirationMichigan Technological UniversityDavid R. Shonnard28 Products from Fermentation Bioprocess Engineering: Basic ConceptsShuler and Kargi, Prentice Hall, 200215 Michigan Technological UniversityDavid R. Shonnard29 Metabolic Engineering (ME) the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions(s) or the introduction of new one(s) with the use of recombinant DNA technology . It is a field that employs the following skills+ Applied molecular biology+ Reaction Engineering+ Systems analysis Metabolic Engineering: Principles and Methodologies Stephanopoulos, Aristidou, and Nielsen, Academic Press, 1998 Michigan Technological UniversityDavid R.

10 Shonnard30 Metabolic pathway Analysis Metabolic Engineering: Principles and Methodologies Stephanopoulos, Aristidou, and Nielsen, Academic Press, 1998 16 Michigan Technological UniversityDavid R. Shonnard31 Principles of ME and Mixed Acid Fermentation Metabolic Engineering: Principles and Methodologies Stephanopoulos, Aristidou, and Nielsen, Academic Press, 1998 1. Rates of intra-cellular reactions can be measured by extra-cellular product accumulation. (ATP)2. The redox balance (balance on NADH consumption and generation) must Technological UniversityDavid R. Shonnard32 Analysis of E. Coli Mixed Acid Fermentation Metabolic Engineering: Principles and Methodologies Stephanopoulos, Aristidou, and Nielsen, Academic Press, 1998 1.