Generating theoretical possible pathways for the production of Lycopene in E.Coli using Retrosynthesis tools

Creative Commons License: CC-BY Questions:
  • Which heterologous pathways are candidates to produce a compound in a chassis?

  • Define what data are required to run RetroSynthesis analysis.

  • Understanding how to run the tools to search heterologous pathways.

Time estimation: 30 minutes
Supporting Materials:
Published: Jun 23, 2022
Last modification: Apr 16, 2024
License: Tutorial Content is licensed under Creative Commons Attribution 4.0 International License. The GTN Framework is licensed under MIT
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Galaxy-SynBioCAD portal is the first toolshed for synthetic biology, metabolic engineering, and industrial biotechnology (Hérisson et al. 2022). It provides a set of Retrosynthesis tools aimed at finding pathways to synthesize heterologous compounds in chassis organisms (RetroRules (Duigou et al. 2018), RetroPath2.0 (Delépine et al. 2018), RP2Paths, rpCompletion).

Retrosynthesis is a concept originally proposed for synthetic chemistry where chemists have to work backwards, starting from a target product to reach precursors that are endogenous to the chassis (host organism).

Typically, the target compound, also named “source compound” is the compound of interest one wishes to produce, while the precursors are usually compounds that are natively present in a chassis strain.

Chassis - target

In this tutorial, we want to obtain the reactions producing the lycopene (source) into the iML1515 Escherichia Coli strain (chassis).

To do that, we will use the following RetroSynthetis Workflow composed of 3 key steps.

First, we aggregate the metabolites present in the chassis and download reaction rules.

Then, RetroPath2.0 generates feasible metabolic routes between a collection of chemical species contained within a GEM SBML (Systems Biology Markup Language) file of the selected organism, a target molecule that the user wishes to produce, and reaction rules extracted from RetroRules.

Lastly, the metabolic network is then deconstructed into individual pathways using RP2paths and rpCompletion takes those individual metabolic pathways to filter them (duplicated pathways are removed), splits them into sub-pathways by adding the appropriate cofactors, and finally converted them to SBML files.

Retrosynthesis Workflow.

Note that we will run the steps of this workflow individually so as not to neglect the understanding of the intermediate steps as well. Then, we will run the workflow automatically so that it itself retrieves the outputs from the previous step and gives them as input to the next tool.


In this tutorial, we will cover:

  1. Data Preparation
    1. Download a model
    2. Create a sink file from the SBML model
    3. Retrieve the reaction rules
  2. Run the Retrosynthesis algorithm
    1. Launch RetroPath2.0
  3. Enumerate pathways with RP2paths
    1. Split the network
    2. Refine reactions
  4. Run the RetroSynthesis Workflow
  5. Conclusion

Data Preparation

RetroSynthesis workflow will be run with the following inputs:

  1. The International Chemical Identifier (InChI) of the compound of interest to produce,
  2. The structure of metabolites present in the chosen chassis (E. coli),
  3. Reaction rules (generated by RRules Parser node that calls RetroRules).

The data used are pretty straight forward to obtain.
Firstly, we download an SBML model, then we select all sinks to use into the RetroPath2.0 software from this model.
Lastly, we request from RetroRules all possible reactions to find a chemical reaction cascade that produces the target.

Download a model

Hands-on: Select a model.
  1. Run Pick SBML Model ( Galaxy version 0.0.1) with the following parameters:
    • galaxy-dropdown “Strain”: Escherichia coli str. K-12 substr. MG1655 (iML1515)
    Comment: What does this tool do?

    The selected SBML model is downloaded from the BiGG database.

  1. What is the file format of the model?
  1. The SBML is based on XML.

Create a sink file from the SBML model

Hands-on: Generate a sink file.
  1. Run Sink from SBML ( Galaxy version 5.12.1) with the following parameters:
    • param-file “Strain”: sbml_model (output of Pick SBML Model tool)
    • “SBML compartment ID”: c
    Comment: Choose a compartment corresponding to your model

    You can specify the compartment from which the tool will extract the chemical species. The default is c, the BiGG code for the cytoplasm. If the user wishes to upload an SBML file from another source, then this value must be changed.

  1. What this tool does?
  2. How many columns are in the file?
  1. This tool creates a friendly CSV file format that can be used as sink input for RetroPath2.0.
  2. Click on galaxy-eye, you should see 2 columns: “Name” and “InChi”

Retrieve the reaction rules

Hands-on: Generate a file with all reactions.
  1. Run RRules Parser ( Galaxy version 2.4.6) with the following parameters:
    • “Rule Type”: retro
    • “Select the diameters of the reactions rules”: 2, 4, 6, 8, 10, 12, 14 and 16
    • galaxy-toggle “Compress output”: no
    Comment: How to choose a right diameter ?

    The diameter of the sphere including the atoms around the reacting center. The higher is the diameter, the more specific are the rules.

  1. Does a low diameter select specific rules?
  2. How many rows are in the file?
  1. No, a low diameter selects more unspecific rules.
  2. More than 200 thousands!

Run the Retrosynthesis algorithm

RetroPath2.0 is an open-source tool for building retrosynthesis networks by combining reaction rules and a retrosynthesis-based algorithm to link the desired target compound to a set of available precursors. The RetroPath2.0 tool is freely available at myExperiment. The retrosynthesis network is outputted as a CSV file providing reactions in the reaction SMILES format and chemicals in both SMILES and InChI formats along with other information like the score for each reaction.

Launch RetroPath2.0

Hands-on: Build a reaction network
  1. Run RetroPath2.0 ( Galaxy version 2.3.0) with the following parameters:
    • param-file “Rules File”: out_rules (output of RRules Parser tool)
    • param-file “Sink File”: sink (output of Sink from SBML tool)
    • “InChI type”: By string
      • “Source InChI”: InChI=1S/C40H56/c1-33(2)19-13-23-37(7)27-17-31-39(9)29-15-25-35(5)21-11-12-22-36(6)26-16-30-40(10)32-18-28-38(8)24-14-20-34(3)4/h11-12,15-22,25-32H,13-14,23-24H2,1-10H3/b12-11+,25-15+,26-16+,31-17+,32-18+,35-21+,36-22+,37-27+,38-28+,39-29+,40-30+

    Be careful, you must have InChi= in front of your InChi key.

  1. What is the file format produce by RetroPath2.0?
  1. A csv file.

Enumerate pathways with RP2paths

The RetroPath2.0 algorithm produces a reaction network, we want to have one pathway per file in SBML format. We need to split the network and perform some adjustments to these pathways.

Split the network

Hands-on: Build a reaction network
  1. Run RP2paths ( Galaxy version 1.5.0) with the following parameters:
    • param-file “RetroPath2.0 Pathways”: Reaction_Network (output of RetroPath2.0 tool)
    Comment: Principle

    Extracts the set of pathways that lies in a metabolic space file output by the RetroPath2.0 workflow

  1. Why producing multiple pathways?
  2. How many outputs are produced?
  1. One network could represent several pathways, so several solutions.
  2. Two outputs are produced: one corresponding to the metabolites, the other one corresponding to the pathways.

Refine reactions

Hands-on: Refine reactions
  1. Run Complete Reactions ( Galaxy version 5.12.2) with the following parameters:
    • param-file “RP2paths pathways”: master_pathways (output of RP2paths tool)
    • param-file “RP2paths compounds”: compounds (output of RP2paths tool)
    • param-file “RetroPath2.0 metabolic network”: Reaction_Network (output of RetroPath2.0 tool)
    • param-file “Sink from SBML”: sink (output of Sink from SBML tool)
    Comment: Principle

    Each reaction rule can correspond to several template reactions, the task here is to enumerate the different possible transformations according to these templates. Because the RetroRules reaction rules consider only one substrate at a time, some compounds are by purpose omitted: the task here is to complete the predicted reactions by putting back these omitted compounds (mostly cofactors). The node converts each predicted pathway to distinct SBML files.

  1. How many solutions are found?
  2. In which file format are the pathways?
  3. Do these pathways are a good solution?
  1. We have 9 candidates.
  2. The pathways are represented in a SBML format.
  3. It’s a trap! We don’t know if they represent a good solution. We need to evaluate them.

Run the RetroSynthesis Workflow

In this section, you can run the RetroSynthesis Workflow more easily and fastly following these instructions:

Hands-on: Execute the entire workflow in one go.
  1. Import the workflow into Galaxy

    Hands-on: Importing and launching a GTN workflow
    Launch RetroSynthesis workflow workflow.

  2. Click on Workflow on the top menu bar of Galaxy. You will see RetroSynthesis workflow.
  3. Click on the workflow-run (Run workflow) button next to your workflow
  4. Provide the workflow with the following parameters:
    • “Target to produce”: Provide the following Inchi source: InChI=1S/C40H56/c1-33(2)19-13-23-37(7)27-17-31-39(9)29-15-25-35(5)21-11-12-22-36(6)26-16-30-40(10)32-18-28-38(8)24-14-20-34(3)4/h11-12,15-22,25-32H,13-14,23-24H2,1-10H3/b12-11+,25-15+,26-16+,31-17+,32-18+,35-21+,36-22+,37-27+,38-28+,39-29+,40-30+
    • “Strain”: Select Escherichia coli str. K-12 substr. MG1655 (iML1515) SBML model.

    All the outputs will be automatically generated and identical to the previous ones.


In this tutorial we produced candidates pathways to produce Lycopene in an Esherichia Coli strain. Three main steps were involved:

  1. Preprocessing data
  2. Run Retrosynthesis algorithm with RetroPath2.0
  3. Enumerate all solutions found by RetroPath2.0