cm4125 bioprocess engineering lab: week 4: introduction to...

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CM4125 Bioprocess Engineering Lab:CM4125 Bioprocess Engineering Lab:Week 4: Introduction to Metabolic Week 4: Introduction to Metabolic

Engineering and Metabolic Flux AnalysisEngineering and Metabolic Flux Analysis

InstructorsInstructors: David R. Shonnard: David R. Shonnard11, Susan T. Bagley, Susan T. Bagley22

Laboratory Teaching AssistantLaboratory Teaching Assistant: Abraham Martin: Abraham Martin--GarciaGarcia

1 Chemical Engineering, 2 Biological Sciences1 Chemical Engineering, 2 Biological SciencesMichigan Technological University, Houghton, MI 49931Michigan Technological University, Houghton, MI 49931

January 31, 2006January 31, 2006102 Chemical Sciences and Engineering Building102 Chemical Sciences and Engineering Building

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Presentation OverviewPresentation Overview

nn Introduction to Metabolic EngineeringIntroduction to Metabolic Engineering

nn An Example of Metabolic Engineering: Ethanol An Example of Metabolic Engineering: Ethanol Production from Production from LignocellulosicLignocellulosic Biomass Using a Biomass Using a GeneticallyGenetically--Engineered Engineered E. coliE. coli. .

nn Metabolic Flux Analysis (MFA)Metabolic Flux Analysis (MFA)

nn Defining the Reaction Defining the Reaction StoichiometryStoichiometry in MFA in MFA

nn Defining the Reaction Rate Equations in MFA Defining the Reaction Rate Equations in MFA

nn SummarySummary

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What is Metabolic Engineering?What is Metabolic Engineering?

��the directed improvement of product formation or the directed improvement of product formation or cellular properties through the modification of specific cellular properties through the modification of specific biochemical reactions or the introduction of new ones biochemical reactions or the introduction of new ones with the use of recombinant DNA technologywith the use of recombinant DNA technology��

The targeting of specific biochemical reactions within the cell The targeting of specific biochemical reactions within the cell for for modification is modification is thethe essential feature of metabolic engineering. To essential feature of metabolic engineering. To achieve this targeting, we use achieve this targeting, we use Metabolic Flux AnalysisMetabolic Flux Analysis (MFA). (MFA).

Stephanopoulos, et al., 1998, Metabolic Engineering: Principles and Methodologies, Academic Press

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Old Practice of Metabolic EngineeringOld Practice of Metabolic Engineering

The concept of manipulating cellular metabolism for The concept of manipulating cellular metabolism for the purpose of increasing production of a desired the purpose of increasing production of a desired product is an old one. product is an old one. MicrooganismsMicrooganisms have been have been developed with enhanced metabolic features. developed with enhanced metabolic features.

�� DNA mutations using chemical treatmentDNA mutations using chemical treatment�� Selection techniques to identify strainsSelection techniques to identify strains�� Notable successesNotable successes

((C. C. glutamicumglutamicum for Lfor L--lysine production, other strains for lysine production, other strains for production of amino acids, antibiotics, solvents, and vitamins.production of amino acids, antibiotics, solvents, and vitamins.

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Modern Practice of Metabolic EngineeringModern Practice of Metabolic Engineering

��The combination of analytical methods to quantify The combination of analytical methods to quantify fluxes and their control with molecular biological fluxes and their control with molecular biological techniques to implement suggested genetic techniques to implement suggested genetic modificamodifica--tionstions is the essence of metabolic engineering.is the essence of metabolic engineering.��

�� Fundamental biochemical pathway studiesFundamental biochemical pathway studies�� Engineering analysis of biochemical reaction pathwaysEngineering analysis of biochemical reaction pathways�� Monitoring of excreted metabolic products (HPLC)Monitoring of excreted metabolic products (HPLC)�� Monitoring of intracellular metabolites and biomassMonitoring of intracellular metabolites and biomass

constituents (using carbon 13 labeled substrates)constituents (using carbon 13 labeled substrates)�� Modern molecular biology techniques Modern molecular biology techniques


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An Example of Metabolic Engineering: An Example of Metabolic Engineering: Ethanol from Ethanol from LignocellulosicLignocellulosic BiomassBiomass

Sustainable Forestry

Logistics

Vehicular Performance

CO2

BiomassProcessing

Photo: Glacial Lakes Energy

Electricity Export and Carbon Credit

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A Shift to BioA Shift to Bio--Based ProductsBased Products

The DOE estimates that US forests can provide 0.10 - 0.40 billion dry tons biomass/yr.

Improved forest management, genetically improved crops, optimized bioprocessing, advanced engine technologies, and hybrid powertrains can be used in an integrated effort to displace 10 - 55% of U.S. gasoline demand (2004).

Harvest energy from the earth’s

surface, and close the cycle for CO2

Earth’s production of plant biomass is 8 times the current consumption of energy.

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BioBio--fuels Benefitsfuels Benefits

�� Lower emission of climate active COLower emission of climate active CO22/mile /mile �� Decreased demand for imported petroleumDecreased demand for imported petroleum�� Job creation and economic diversificationJob creation and economic diversification

For a 50 million gallon biomass ethanol / year facilityFor a 50 million gallon biomass ethanol / year facility+ + ~~ $100 million / year from sale of ethanol$100 million / year from sale of ethanol+ + Stimulate $200 million / year in the local economyStimulate $200 million / year in the local economy+ + ~~190 new long190 new long--term jobs + 450 spinterm jobs + 450 spin--off jobs off jobs + + 500 construction jobs for 2 years500 construction jobs for 2 years

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Process to Convert Process to Convert CellulosicCellulosic Biomass to Biomass to EthanolEthanol

1 –3 mm

0.8% H2SO4160ºC10 min

Trichodermareeseicellulases

Genetically-engineered E. coli


or process heat and power

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Composition of Dry Composition of Dry CellulosicCellulosic BiomassBiomass

Cellulose(35-50%)

Dry CellolosicBiomass

Hemicellulose(20-35%)

Glucose6-C sugars

Lignin(12-20%)

XyloseArabanoseMannoseGalactose(5-C sugars)

hydrolysishydrolysis

no hydrolysis

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The Challenge of Fermenting all Sugars The Challenge of Fermenting all Sugars in in LignocellulosicLignocellulosic BiomassBiomass

Saccharomycescervisiae

Zymomonasmobilis

Ferment glucose to ethanolUtilize 6C sugars onlyTolerant to ethanol

Can these microorganisms be genetically engineered to utilize 5C sugars?

Escherichia coli

Can not ferment glucose to ethanolCan utilize 6C and 5C sugars

Is it easier to genetically engineer E. coli to ferment ethanol?

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The Challenge of Diverting all Sugars to The Challenge of Diverting all Sugars to EthanolEthanol Stephanopoulos, et al., 1998, Metabolic Engineering:

Principles and Methodologies, Academic Press

There is a competition for pyruvate among different pathways. The pathway containing the highest concentration of enzymes and where these enzymes have the highest activity (Vmax) and affinity (Km) will have the highest flux of metabolites and divert the majority of the substrate to endproduct.

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Genetically Engineer This Pathway into Genetically Engineer This Pathway into E. coliE. coli

Two genes are needed. One for pyruvatedecarboxylase and another for alcohol dehydrogenase. These enzymes working together in the cell will divert Pyruvate away from other fermenta-tion products to ethanol. This would convert E. coli into an ethanol-producing microorganism, where before it was not!

“Principles of Biochemistry”, Lehninger, Worth

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Genetic Engineering of Ethanol Genetic Engineering of Ethanol Production in Production in E. coliE. coli

A plasmid for Pyruvatedecarboxylase (pdc)

Ingram, Conway, Clark, Sewell, and Preston,“Genetic engineering of ethanol production in E. coli”,App. Environ. Microbio., 1987, 53(10), 2420-2425.

A plasmid for Alcohol dehydrogenase (adh)

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Ethanol Production Ethanol Production in Sealed Cultures in Sealed Cultures of of E. coliE. coli TC4TC4

Ingram, Conway, Clark, Sewell, and Preston,“Genetic engineering of ethanol production in E. coli”,App. Environ. Microbio., 1987, 53(10), 2420-2425.

High Performance Liquid Chromatography Profiles

G = glucoseS = succinateL = lactic acidA = acetic acidU = unknownE = ethanol

Plasmid-free TC4 TC4withpLOI295

TC4withpLOI284

TC4withpLOI276

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More Results for Ethanol Production in More Results for Ethanol Production in GeneticallyGenetically--Modified Modified E. coliE. coli


Using a different recombinant strain of E. coli, the Ingram group obtained these results under both aerobic and anaerobic conditions. Wild type E. coli metabilizes pyruvate through PDH and PFL yielding primarily cell growth, acetate and CO2. In the recombinant strain under aerobic conditions, significant ethanol is produced because the Km for heterologous PDC from Z. mobilis is comparable to endogenous PDH and much lower than for endogenous LDH. Furthermore, Km for heterologous ADH II from Z. mobilis is about a factor of 4 lower than Km for endogenous NADH oxidase, thus effectively diverting pyruvate to ethanol. Under anaerobic conditions, again Km for PDC and ADH II are significant lower than for LDH and PFL, and the apparent Km of the native enzymes involved in NAD+ regeneration are higher than for Z. mobilis ADH II.

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A Comparison of Enzyme Properties in A Comparison of Enzyme Properties in GeneticallyGenetically--Modified Modified E. coliE. coli


“Overall, overexpressed ethanologenic Z. mobilis enzymes in E. coli are quite competitive with respect to the native enzymes in channeling carbon (pyruvate) and reducing power (NADH) to ethanol. “

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Metabolic Flux Analysis (MFA)Metabolic Flux Analysis (MFA)

The purpose of MFA is to describe with the minimum The purpose of MFA is to describe with the minimum number of equations the flow of material through a number of equations the flow of material through a cellular metabolic network. This requires that the set cellular metabolic network. This requires that the set of of stoichiometricstoichiometric and/or reaction rate equations be and/or reaction rate equations be solved for the concentrations of solved for the concentrations of intracellular metabolic intracellular metabolic intermediates and/or pathway fluxesintermediates and/or pathway fluxes using measured using measured values of values of extracellularextracellular metabolic productsmetabolic products. .

We start with a general We start with a general stoichiometrystoichiometry for cellular for cellular reactions. reactions.

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StoichiometryStoichiometry of Cellular Reactionsof Cellular Reactions

Consider a network with Consider a network with NN substrates that are converted to substrates that are converted to MMmetabolic products (metabolic products (extracellularextracellular and measurable) and and measurable) and QQ biomass biomass constituents (DNA, protein, lipids constituents (DNA, protein, lipids �� also measurable). The also measurable). The network consists of network consists of JJ reactions involving reactions involving KK intracellular intracellular metabolites (not measurable, but predicted). A mole balance on metabolites (not measurable, but predicted). A mole balance on the the jthjth cellular reactioncellular reaction yieldsyields

where subscript where subscript ii refers to each substrate, product or metabolite, refers to each substrate, product or metabolite, SS is substrate, is substrate, PP is is extracellularextracellular product, product, XXmacromacro is a biomass is a biomass constituent, and constituent, and XXmetmet is a pathway intermediate. is a pathway intermediate.

∑∑∑∑====

=+++K

iimetji

Q

iimacroji

M

iiji

N

iiji XgXPS

1,

1,

11

0γβα

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StoichiometryStoichiometry of Cellular Reactions (cont.)of Cellular Reactions (cont.)

In the metabolic model there will be an equation like the one onIn the metabolic model there will be an equation like the one onthe previous slide for each of the the previous slide for each of the JJ cellular reactions. It is cellular reactions. It is convenient to write the convenient to write the stoichiometrystoichiometry for all J reactions in a for all J reactions in a compact form using compact form using matrix notationmatrix notation. .

where where AA, , BB, , FF, and , and GG are matrices containing are matrices containing stoichiometricstoichiometriccoefficients in the coefficients in the JJ reactions for the substrates, products, reactions for the substrates, products, biomass constituents, and pathway intermediates, respectively. biomass constituents, and pathway intermediates, respectively.

0 GX X BP AS =+Γ++ metmacro

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Some Rules in Defining Metabolic ReactionsSome Rules in Defining Metabolic Reactions

1.1. Only intracellular metabolites at Only intracellular metabolites at branch pointsbranch points in the in the metabolic pathway are included in the set of reactions.metabolic pathway are included in the set of reactions.

2.2. Reactions on Reactions on linear pathwayslinear pathways between branch points are not between branch points are not included since all of these reactions proceed at the same rate included since all of these reactions proceed at the same rate at steady state. at steady state.

3.3. All metabolites along a linear pathway may be All metabolites along a linear pathway may be lumpedlumped into an into an overall reaction for that linear pathway. overall reaction for that linear pathway.

4.4. Include all substrates and products in the set of reactions. Include all substrates and products in the set of reactions. 5.5. Reactants are assigned negative Reactants are assigned negative stoichiometricstoichiometric coefficients.coefficients.6.6. Reaction products are assigned positive coefficients. Reaction products are assigned positive coefficients.

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An Example of An Example of StoichiometryStoichiometry::Mixed Acid Fermentation by Mixed Acid Fermentation by E. coliE. coli


In mixed fermentation, seven products are produced from the uptake of glucose

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Pathway Reactions Pathway Reactions Mixed Acid Fermentation by Mixed Acid Fermentation by E. coliE. coli

0 ethanol 2NADH -CoA -acetyl- 8.

0 ATP acetate CoA -acetyl- 7.

0 H CO formate- 6.

0 formate CoA -acetyl pyruvate - 5.

0 lactate NADH - pyruvate- 4.

0 ATP pyruvate PEP- 3.

0 succinate 2NADH - CO - PEP- 2.

0 NADH PEP glucose2

1- 1.

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2

=+=++

=++=++

=+=++

=+

=++

In mixed fermentation, eight metabolic reactions define the reaction network. A total of five intracellular metabolites are also included. Notice that no biomass constituents are included because no anabolic reactions are involved and only fueling reactions are included. With a total of 13 chemicals and only 8 reactions,

the degrees of freedom for this problem is 5, thus atleast 5 concentrations must be measured in order to determine the other intracellular metabolites and extracellular products.

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ATP ATP StoichiometryStoichiometry::Mixed Acid Fermentation by Mixed Acid Fermentation by E. coliE. coli



0 H CO formate- 6.





0 NADH PEP glucose2

1- 1.

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2

=+=++

=++=++

=+=++

=+

=++


The fourth column of the G matrix contains the stoichiometric coefficients for ATP metabolism under anaerobic conditions. The third and seventh reactions in the network are responsible for ATP production. Intracellular ATP production can be measured directly by monitoring acetate production and the difference between glucose uptake and succinate production. Thus, intracellular metabolite flux can be measured by monitoring extracellular products; a key foundation of MFA. Knowing intracellular ATP provides an estimate of ATP consumption rate for cell growth and maintenance.

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NADH NADH StoichiometryStoichiometry::Mixed Acid Fermentation by Mixed Acid Fermentation by E. coliE. coli



0 H CO formate- 6.





0 NADH PEP glucose2

1- 1.

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2

=+=++

=++=++

=+=++

=+

=++


The redox balance (NADH balance) has to close for the conversion of sugar to different fermentation products, the matrix stoichiometric equations indicate that the uptake of glucose must be related to the formation of succinate, lactate, and ethanol.

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NADH Balance in Anaerobic FermentationNADH Balance in Anaerobic FermentationMixed Acid Fermentation by Mixed Acid Fermentation by E. coliE. coli

consumed! glucose moles 100

per produced NADH moles 200 matcheswhich

200.5 49.8 2 79.5 10.7 2

tryStoichiome from Determined Consumed NADH

=×++×


The redox balance (NADH balance) has to close for the conversion of sugar to different fermentation products. The matrix stoichiometric equations indicate that the uptake of glucose must be related to the formation of succinate (rxn 2), lactate (rxn. 4), and ethanol (rxn. 8).

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Rates of Cellular ReactionsRates of Cellular Reactions

In MFA, the In MFA, the ratesrates of enzyme catalyzed reactions in metabolic pathways is of enzyme catalyzed reactions in metabolic pathways is of even greater importance than of even greater importance than stoichiometrystoichiometry. The purpose of . The purpose of metabolic engineering is to alter the flux of material through mmetabolic engineering is to alter the flux of material through metabolic etabolic pathways, and therefore the ability to pathways, and therefore the ability to predict intracellular fluxespredict intracellular fluxes, and , and more importantly, to more importantly, to identify enzymatic reactions to modifyidentify enzymatic reactions to modify is a primary is a primary objective. objective.

The The net specific uptake rate for the net specific uptake rate for the ithith substratesubstrate is the sum of its is the sum of its consumption rate in al consumption rate in al JJ reactions. reactions.

where subscript where subscript jj refers to each reaction in the network and refers to each reaction in the network and ννjj is the rate is the rate of the of the jthjth reaction in the network. reaction in the network.

∑=

=J

jjjiisr

1, να

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Rates of Cellular Reactions (cont.)Rates of Cellular Reactions (cont.)

Similarly, the net specific rate of formation of the Similarly, the net specific rate of formation of the ithith metabolic metabolic productproduct is is

For the biomass constituents and intracellular metabolites we caFor the biomass constituents and intracellular metabolites we can n write write

We can write these summation equationWe can write these summation equationcompactly in matrix notation. compactly in matrix notation.

∑=

=J

jjjiipr

1, νβ

∑=

=J

jjjiimacror

1, νγ ∑

=

=J

jjjiimet gr

1, ν


The matrix equations are a compact way to show that a vector of net component fluxes (r) is equal to a matrix multiplied by a vector of reaction rates (v), where the coefficients in these matrices are the transpose of the elements in the stoichiometric matrices (each row of the stoichiometeric matrix is situated in the equivalent column of the transposed matrix). We wish in fact to determine the vector of reaction rates in order to solve for the metabolic fluxes in the reaction network.

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Rates of Cellular Reactions:Rates of Cellular Reactions:Mixed Acid Fermentation by Mixed Acid Fermentation by E. coliE. coli

The The specific glucose uptake ratespecific glucose uptake rate is given by the matrix is given by the matrix equation equation

Thus the flux from glucose to PEP, which is given by Thus the flux from glucose to PEP, which is given by νν11, is 2 times the specific glucose uptake rate. , is 2 times the specific glucose uptake rate.


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Rates of Cellular Reactions: (cont.)Rates of Cellular Reactions: (cont.)Mixed Acid Fermentation by Mixed Acid Fermentation by E. coliE. coli

For the five intracellular metabolites we state these five rate For the five intracellular metabolites we state these five rate equations as,equations as,

At steady state, many of the rates on the left hand side are zerAt steady state, many of the rates on the left hand side are zero. o. The measurements of The measurements of extracellularextracellular products and biomass products and biomass components will allow for the prediction of these fluxes. components will allow for the prediction of these fluxes.


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Summary of ME and MFA ConceptsSummary of ME and MFA Concepts

nn Metabolic Engineering is a new and powerful approach to Metabolic Engineering is a new and powerful approach to understand cellular metabolism and then improve production of understand cellular metabolism and then improve production of desired products from microorganisms.desired products from microorganisms.

nn New microorganisms have been created with enhanced New microorganisms have been created with enhanced metabolic features that allows for efficient production of desirmetabolic features that allows for efficient production of desired ed products from microbial fermentations (Ethanol from products from microbial fermentations (Ethanol from LignocellulosicLignocellulosic biomass example)biomass example)

nn Metabolic Flux Analysis (MFA) allows for prediction of Metabolic Flux Analysis (MFA) allows for prediction of intracellular reaction fluxes using a set of metabolic reactionsintracellular reaction fluxes using a set of metabolic reactions and and measurements of measurements of extracellularextracellular products. products.

cm4125 bioprocess engineering lab: week 4: introduction to...

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