Team:TU Delft/Modeling/MFA/additional pathways

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Revision as of 10:17, 24 October 2010

Pathways added to the E. coli metabolic network

Figure 1 – Reaction taken from [http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION-IN-PATHWAY&object=NADH-DEHYDROG-A-RXN 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 [http://www.mpi-magdeburg.mpg.de/projects/cna/cna.html here].

There was one change made in the standard network 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.

Alkane degradation

To link alkanes to the existing network, the beta-oxidation was chosen as entry point. Several genes were used to transform alkanes in to alkanoic acid which enters the beta oxydation cycle. These genes were:

AlkB2 (EC 1.14.15.3)

Figure 2 – Reaction taken from [http://metacyc.org/META/new-image?type=REACTION&object=ALKANE-1-MONOOXYGENASE-RXN Metacyc]

Reaction for CNA; n-alkane + reduced rubredoxin + O2 -> n-alkanol + oxidized rubredoxin

RubA3/RubA4 (EC 1.18.1.1)

Figure 3 – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RUBREDOXIN--NAD%2b-REDUCTASE-RXN Metacyc]

Reaction for CNA; oxidized rubredoxin + NADH -> reduced rubredoxin

ADH (EC 1.1.1.1)

Figure 4 – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=ALCOHOL-DEHYDROG-GENERIC-RXN Metacyc]

Reaction for CNA; n-alkanol -> n-aldehyde + NADH

ALDH (EC 1.2.1.3)

Figure 5 – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=RXN-4142 Metacyc]

Reaction for CNA; n-aldehyde -> n-fatty acid acid + NADH

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.

fatty acyl-CoA synthetase (EC 6.2.1.3)

Figure 6 – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=ACYLCOASYN-RXN Ecocyc]

Reaction for CNA; n-fatty acid + ATP -> n-saturated fatty acyl-CoA + AMP

Adenylate kinase (EC 2.7.4.3)

Figure 7 – Reaction taken from [http://biocyc.org/META/NEW-IMAGE?type=REACTION&object=ADENYL-KIN-RXN Ecocyc]

Reaction for CNA; 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 [http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=FAO-PWY&detail-level=2 Ecocyc]

Reaction for CNA; n-saturated fatty acyl-CoA -> (n - 2)-saturated fatty acyl-CoA + acetyl-CoA + FADH2 + NADH

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 oxidative 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 [http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION-IN-PATHWAY&object=NADH-DEHYDROG-A-RXN Ecocyc]

[http://biocyc.org/ECOLI/NEW-IMAGE?type=PATHWAY&object=PWY0-1335 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 [http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION&object=RXN0-6369 (EC 1.7.99.4)]

Figure 10 – Reaction taken from [http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION-IN-PATHWAY&object=NADH-DEHYDROG-A-RXN 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 [http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION-IN-PATHWAY&object=NADH-DEHYDROG-A-RXN Ecocyc]

The pathway is displayed here [http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY1-3&detail-level=2 (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 http://biocyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY-6270&detail-level=2

Figure 12 – Reaction taken from [http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION-IN-PATHWAY&object=NADH-DEHYDROG-A-RXN Ecocyc]

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 http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION&object=FHLMULTI-RXN

Figure 13 – Reaction taken from [http://biocyc.org/ECOLI/NEW-IMAGE?type=REACTION-IN-PATHWAY&object=NADH-DEHYDROG-A-RXN Ecocyc]

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

Formate + H+ -> H2 + CO2

hydrogen export

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