Team:TU Delft/Modeling/MFA/additional pathways

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(Difference between revisions)
(Alkane degradation)
(Alkane degradation)
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==Alkane degradation==
==Alkane degradation==
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[[Image:Team_TUDelft_AlkB.png|thumb|270px|right|'''Figure 2''' – Reaction taken from [http://metacyc.org/META/new-image?type=REACTION&object=ALKANE-1-MONOOXYGENASE-RXN Metacyc]]]
 
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[[Image:Team_TUDelft_EC1672.png|thumb|270px|right|'''Figure 3''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RUBREDOXIN--NAD%2b-REDUCTASE-RXN Metacyc]]]
 
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[[Image:Team_TUDelft_ADH.png|thumb|270px|right|'''Figure 4''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=ALCOHOL-DEHYDROG-GENERIC-RXN Metacyc]]]
 
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[[Image:Team_TUDelft_ALDH.png|thumb|270px|right|'''Figure 5''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RXN-4142 Metacyc]]]
 
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[[Image:Team_TUDelft_fadK.png|thumb|270px|right|'''Figure 6''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=ACYLCOASYN-RXN Ecocyc]]]
 
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[[Image:Team_TUDelft_adk.png|thumb|270px|right|'''Figure 7''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=ADENYL-KIN-RXN Ecocyc]]]
 
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[[Image:Team_TUDelft_fa_oxy.png|thumb|270px|right|'''Figure 8''' – Reaction taken from [http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=FAO-PWY&detail-level=2 Ecocyc]]]
 
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'''AlkB2 (EC 1.14.15.3)'''
'''AlkB2 (EC 1.14.15.3)'''
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[[Image:Team_TUDelft_AlkB.png|thumb|270px|right|'''Figure 2''' – Reaction taken from [http://metacyc.org/META/new-image?type=REACTION&object=ALKANE-1-MONOOXYGENASE-RXN Metacyc]]]
The reaction for AlkB2 is shown in figure 2.
The reaction for AlkB2 is shown in figure 2.
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'''RubA3/RubA4 (EC 1.18.1.1)'''
'''RubA3/RubA4 (EC 1.18.1.1)'''
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[[Image:Team_TUDelft_EC1672.png|thumb|270px|right|'''Figure 3''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RUBREDOXIN--NAD%2b-REDUCTASE-RXN Metacyc]]]
The reaction for the regeneration of rubredoxin is shown in figure 3.
The reaction for the regeneration of rubredoxin is shown in figure 3.
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'''ADH (EC 1.1.1.1)'''
'''ADH (EC 1.1.1.1)'''
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[[Image:Team_TUDelft_ADH.png|thumb|270px|right|'''Figure 4''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=ALCOHOL-DEHYDROG-GENERIC-RXN Metacyc]]]
The reaction for ADH is shown in figure 4.
The reaction for ADH is shown in figure 4.
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'''ALDH (EC 1.2.1.3)'''
'''ALDH (EC 1.2.1.3)'''
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[[Image:Team_TUDelft_ALDH.png|thumb|270px|right|'''Figure 5''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RXN-4142 Metacyc]]]
The reaction for ALDH is shown in figure 5.
The reaction for ALDH is shown in figure 5.
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'''fatty acyl-CoA synthetase (EC 6.2.1.3)'''
'''fatty acyl-CoA synthetase (EC 6.2.1.3)'''
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[[Image:Team_TUDelft_fadK.png|thumb|270px|right|'''Figure 6''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=ACYLCOASYN-RXN Ecocyc]]]
From here the genes are already present in the ''E. coli'' genome. They were not yet present in the metabolic network for ''E. coli'' in CellNetAnalyzer however. So the following reactions and pathways were also implemented in CNA. The reaction for fatty acyl-CoA synthetase is shown in figure 6.
From here the genes are already present in the ''E. coli'' genome. They were not yet present in the metabolic network for ''E. coli'' in CellNetAnalyzer however. So the following reactions and pathways were also implemented in CNA. The reaction for fatty acyl-CoA synthetase is shown in figure 6.
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'''Adenylate kinase (EC 2.7.4.3)'''
'''Adenylate kinase (EC 2.7.4.3)'''
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[[Image:Team_TUDelft_adk.png|thumb|270px|right|'''Figure 7''' – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=ADENYL-KIN-RXN Ecocyc]]]
CNA did not have a reaction to regenerate AMP yet, so the reaction for adenylate kinase was added to the network. The reaction for adenylate kinase is shown in figure 7.
CNA did not have a reaction to regenerate AMP yet, so the reaction for adenylate kinase was added to the network. The reaction for adenylate kinase is shown in figure 7.
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'''Fatty acid beta-oxidation cycle (EC 1.3.99.3    EC 4.2.1.17    EC 1.1.1.35    EC 2.3.1.16)'''
'''Fatty acid beta-oxidation cycle (EC 1.3.99.3    EC 4.2.1.17    EC 1.1.1.35    EC 2.3.1.16)'''
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[[Image:Team_TUDelft_fa_oxy.png|thumb|270px|right|'''Figure 8''' – Reaction taken from [http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=FAO-PWY&detail-level=2 Ecocyc]]]
The reactions for this pathway are shown in figure 8.
The reactions for this pathway are shown in figure 8.

Revision as of 12:09, 24 October 2010

Pathways added to the E. coli metabolic network

Figure 1 – Reaction taken from Ecocyc

The E. coli network from Cell Net Analyzer contains, glycolysis, TCA cycle, pentose phosphate pathway, gluconeogenesis, anapleorotic routes, oxydative phosphorilization and biosynthesis pathways. CellNetAnalyzer can be found here.


There was one change made in the network provided by CellNetAnalyzer for the NADH dehydrogenase reaction, annotated as NADHdehydro in CellNetAnalyzer. In the network of CellNetAnalyzer this reaction exports two protons, but this was changed to 4 as given by Ecocyc.


Alkane degradation

To link alkanes to the existing network, the beta-oxidation was chosen as entry point. Several genese from biobricks were used to transform alkanes in to fatty acids which enter the beta oxydation cycle. These genes were:


AlkB2 (EC 1.14.15.3)

Figure 2 – Reaction taken from Metacyc

The reaction for AlkB2 is shown in figure 2.

The reaction was implemented in CNA as;

n-alkane + reduced rubredoxin + O2 -> n-alkanol + oxidized rubredoxin


RubA3/RubA4 (EC 1.18.1.1)

Figure 3 – Reaction taken from Metacyc

The reaction for the regeneration of rubredoxin is shown in figure 3.

The reaction was implemented in CNA as;

oxidized rubredoxin + NADH -> reduced rubredoxin


ADH (EC 1.1.1.1)

Figure 4 – Reaction taken from Metacyc

The reaction for ADH is shown in figure 4.

The reaction was implemented in CNA as;

n-alkanol -> n-aldehyde + NADH


ALDH (EC 1.2.1.3)

Figure 5 – Reaction taken from Metacyc

The reaction for ALDH is shown in figure 5.

The reaction was implemented in CNA as;

n-aldehyde -> n-fatty acid + NADH


fatty acyl-CoA synthetase (EC 6.2.1.3)

Figure 6 – Reaction taken from Ecocyc

From here the genes are already present in the E. coli genome. They were not yet present in the metabolic network for E. coli in CellNetAnalyzer however. So the following reactions and pathways were also implemented in CNA. The reaction for fatty acyl-CoA synthetase is shown in figure 6.

The reaction was implemented in CNA as;

n-fatty acid + ATP -> n-saturated fatty acyl-CoA + AMP


Adenylate kinase (EC 2.7.4.3)

Figure 7 – Reaction taken from Ecocyc

CNA did not have a reaction to regenerate AMP yet, so the reaction for adenylate kinase was added to the network. The reaction for adenylate kinase is shown in figure 7.

The reaction was implemented in CNA as;

ATP + AMP -> 2 ADP


Fatty acid beta-oxidation cycle (EC 1.3.99.3 EC 4.2.1.17 EC 1.1.1.35 EC 2.3.1.16)

Figure 8 – Reaction taken from Ecocyc

The reactions for this pathway are shown in figure 8.

The lumped reaction was implemented in CNA as;

n-saturated fatty acyl-CoA -> (n - 2)-saturated fatty acyl-CoA + acetyl-CoA + FADH2 + NADH


FADH2 regeneration

The cofactor FADH2 is not yet defined in the network of CNA, so a reaction had to be introduced to regenerate it. FADH2 gives its electrons to ubiquinol just like NADH, however in this process no protons are exported.

The reaction was implemented in CNA as;

1 FADH2 -> 1 QH2


Odd numbered alkanes

For all even numbered alkanes the above reactions completely link them to the main network in CNA. All the cofactors have regeneration reactions and all the alkanes are converted into acetyl-CoA. The final reaction in the beta oxidation cycle (n = 4) produces then two acetyl-CoA. For odd numbered alkanes the final reaction however (n = 5), a propionyl-CoA is generated;

n-saturated fatty acyl-CoA -> propionyl-CoA + acetyl-CoA + FADH2 + NADH


2-methylcitrate cycle (EC 2.3.3.5 EC 4.2.1.79 EC 4.2.1.99 EC 4.1.3.30)

Figure 9 – Reaction taken from Ecocyc

This propionyl-CoA still needs to be linked to the main network in CNA. The pathway that was used to process this metabolite, was a part of the 2-methylcitrate cycle. The reactions for this pathway are shown in figure 9.

The lumped reaction was implemented in CNA as;

propionyl-CoA + oxaloacetic acid -> succinate + pyruvate

Biomass formation

The biomass is formed by many anabolic reactions that make monomers. All the anabolic reactions start at the so called key metabolites. There are 12 key metabolites and they are all in the glycolytic pathway and the TCA cycle. In the tool they are the red metabolites.

NO3 as electron acceptor

In oily environments oxygen diffuses more difficult into the water phase. The oxygen is used for the oxydative phosphorylation, regenerating NADH, and for the first step in the hydrocarbon degradation. To be more efficient with oxygen an additional electron acceptor was introduced.

Figure 9 – Reaction taken from Ecocyc

The standard oxydative phosphorylation (EC 1.6.5.3 EC 1.10.2.-)


The second step will be disabled in the network and be replaced with a nitrate reductase (EC 1.7.99.4)

Figure 10 – Reaction taken from Ecocyc
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The reaction for CNA will be;

NO3- + QH2 -> NO2-

This reaction uses NO3 as an electron acceptor to regenerate NADH and export protons to generate ATP. Less protons are exported per mol of NADH, so the ATP/NADH ratio will drop compared to oxygen. The goal of implementing this pathway however, is to see how much the oxygen requirement of E. coli can be reduced.

PHB production

In previous situations the hydrocarbons were degraded only to form biomass and CO2. It is interesting to see how much product could be made from hydrocarbons. PHB is a polymer of polyhydroxybutyrate. The production pathway of PHB is well known. PHB is a solid product which is to recover in the down stream process.

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Figure 11 – Reaction taken from Metacyc

The pathway is displayed here (EC 2.3.19 EC 1.1.1.36 EC 2.3.1.-)


In this scenario the lumped PHB production pathway was added to metabolic network;

2 acetyl-CoA + NADPH -> (R)-3-hydroxybutanoyl-CoA

the polymerization reaction just consumes (R)-3-hydroxybutanoyl-CoA.

Isoprene production

In previous situations the hydrocarbons were degraded only to form biomass and CO2. It is interesting to see how much product could be made from hydrocarbons. Isoprene is a volatile product found in plants. E. coli will not be able to produce is in the near future, but it is an interesting product It is a very reduced product, with a similar amount of electron per carbon atom. Hydrocarbons have 6 - 6.3 electrons per carbon atom depending on the length and isoprene has 5.6 electron per carbon electron. If these values are close to each other, it has a positive influence on the maximal theoretical yield. Also the volatile nature of isoprene is very favorable for the downstream process

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The pathway is displayed here

Figure 12 – Reaction taken from Metacyc

In this scenario the lumped isoprene production pathway was added to metabolic network;


1 pyruvate + 1 D-glyceraldehyde-3-phosphate + 1 NADPH + 3 NADH + 3 ATP -> isoprene + CO2

isoprene export

Hydrogen production

In previous situations the hydrocarbons were degraded only to form biomass and CO2. It is interesting to see how much product could be made from hydrocarbons. Hydrogen is considered a green fuel. Hydrogen is a volatile product and is easily separated from fermentation broth. It does however contain no carbon atoms, so hydrogen will result in production of CO2 and biomass.


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The pathway is displayed here

Figure 13 – Reaction taken from Ecocyc

In this scenario the hydrogen production pathway was added to metabolic network;


Formate + H+ -> H2 + CO2

hydrogen export


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