Practical 5

Building a model from databases

Our aim is to construct a model of the Entner-Doudoroff pathway of glycolysis in the natural ethanol-fermenting organism Zymomonas mobilis. For examples of how such a model can be exploited, you can find two articles here and here.

Within ScrumPy, there is a module PyoCyc that can extract the required reaction equations from a downloaded copy of the MetaCyc database, starting from a list of the enzyme EC numbers. However, the BioCyc set of databases, including MetaCyc, have moved to a subscription model that makes it more difficult for us to share this with you. Hence you will need to consult the web version of the database and write the reaction equations your self.

Your task is to get the model to work so there is a viable route from glucose to ethano.

Step 1

Download, by right-clicking on its link, the ScrumPy file Zymomonas.spy and save it in the directory you are going to use for this exercise.
Open the model file with ScrumPy. Two editor windows will open. The one for Zymomonas.spy contains some generic components of the model here that we are giving you as a starting point:
1. An Include directive to the file !ZmED.spy which opens as the second, initially empty window into which you are going to write the pathwy reactions.
2. A statement that WATER and PROTONS are regarded as external, freely-available species. Balancing every last PROTON is hard, so we're not going to struggle with t_hat yet.
3. A set of transporter definitions that state that glucose (GLC), ethanol (ETOH) and carbon dioxide (CARBON-DIOXIDE) can exchange between the metabolism and the environment.
4. A generic ATPase reaction that states that any ATP generated will be hydrolysed by other reactions in the cell.
Note that this means you will need to use the same metabolite names for H₂O, H⁺, etc.

Step 2

This list of EC numbers covers the Entner-Doudoroff pathway:EC-2.7.1.2', 'EC-3.1.1.31', 'EC-4.2.1.12', 'EC-4.1.2.14','EC-1.1.1.44', 'EC-1.2.1.12', 'EC-2.7.2.3', 'EC-5.4.2.11', 'EC-4.2.1.11', 'EC-2.7.1.40', 'EC-4.1.1.1', 'EC-1.1.1.1']
You can see the set of reactions associated with each EC number with the following code:
```
for e in ecs:
        for r in db[e]:
                print e, "\n", r.AsScrumPy()
```
Here, each EC number in turn is used as a key, e into the database, which returns a set of reaction records. The method AsScrumPy formats the record as required for the input file.
You need to generate an input file though, so enter the following:
```
spy=open("ZmED.spy","w")
spy.write('# Entner Duodoroff pathway\n')
for e in ecs:
    str = "# " + e +"\n"
    for r in db[e]:
            spy.write(str)
            spy.write(r.AsScrumPy())

spy.close()
```
- After opening the file, ZmED.spy, the first write statement puts an identifying comment in. Then for each EC number, a comment is inserted giving the EC number, since this often doesn't appear in the reaction name.

Step 3

Un-comment the Include directive in the Zymomonas.spy file to load your ZmED.spy and recompile the model using the drop-down ScrumPy menu option. Find out if there is a potential pathway through it by calculating the null space of the stoichiometry matrix:
```
ns = m.sm.NullSpace()
ns
```
- Two windows should now be open, one for Zymomonas.spy and one for ZmED.spy. However, the null space is empty, in other words the system defined by this model cannot support a stead-state flux in any of its reactions.
The common possibilities for the lack of a viable route where there should be one are either missing reactions, or, when all the reactions are present, inconsistencies in metabolite definitions break the connectivity. Looking for dangling (orphan) metabolites can point to both of these issues:
```
m.sm.OrphanMets()
```
If the network connectivity is being broken by inconsistent metabolite naming, edit ZmED.spy, save it, and recompile the model. Then check the null space and orphan metabolites again.
Once you get one or more null space vectors, you can compute the elementary modes of the model and the stoichiometry of their overall reaction to check whether you have the exected conversion of glucose to ethanol.
The model can now be reduced by removing reactions that will always be dead. Calculate the enzyme subsets of the model; all the dead reactions will be in the "dead" subset. Rather than deleting the reactions immediately, remove them from the model by placing a comment marker, "#", at the start of each line belonging to an unwanted reaction. Recompile the model and check that you have not broken the network.
How many reactions are left in ZmED.spy when you have inactivated all the dead ones?

Step 4

Time permitting, go to the next part.