The Subliminal Toolbox: automating steps in the

reconstruction of metabolic networks

Neil SwainstonManchester Centre for Integrative Systems Biology

1st Conference on Constraint-based Reconstruction and AnalysisReykjavik, Iceland

24 June2011

Automation

• Many of these steps can be automated• Subliminal Toolbox

• How much of the reconstruction process can be aided by publicly available resources?• Manual curation still necessary• BUT reduce what needs to be done

• Relies on semantic model annotations• e.g. ChEBI, KEGG terms for metabolites• UniProt terms for enzymes

Model annotations

• Model annotations provide unambiguous, unique identifiers for model components• Not very interesting

• But – also provide link to chem/bioinformatics resources via web services• Automatically extract data through web services• Potentially more interesting / useful

Publicly available resources

Formula,Charge,Structure

GPRrelationships

Compartmentdata

Sequence,Metadata

Web services

Subliminal Toolbox

Protonate

• ChEBI provides web service access to structural data• InChI, SMILES strings

• Cheminformatics software (ChemAxon) can be used to predict charge state at given pH• Updates FORMULA, CHARGE, metabolite name and

annotation• Consistency – facilitates merging

✓✗

Balance

• Reaction elemental and charge balancing• Prevents mass violation and stoichiometric

inconsistencies

• Reactions in resources don’t always balance• Incorrect stoichiometry• Missing protons, water, etc.

• Solution: extract formulae and charges and apply linear programming

Balance

carbon dioxide + 2-Acetolacetate Pyruvate

Ab = 0

A =

Reactants Products Optional reactants Optional productsCO2 C5H7O4 C3H3O3 H+ H20 H+ H20 CO2

C 1 5 -3 0 0 0 0 -1O 2 4 -3 0 1 0 -1 -2H 0 7 -3 1 2 -1 -2 0charge 0 -1 1 1 0 -1 0 0

bminT 1 1 1 0 0 0 0 0

b represents a vector of stoichiometries

CO2 + C5H7O4- C3H3O3

-

Balance

• Linear programming solver solves Ab = 0

Reactants Products Optional reactants Optional productsCO2 C5H7O4 C3H3O3 H+ H20 H+ H20 CO2

C 1 5 -3 0 0 0 0 -1O 2 4 -3 0 1 0 -1 -2H 0 7 -3 1 2 -1 -2 0charge 0 -1 1 1 0 -1 0 0

bminT 1 1 1 0 0 0 0 0

bT 1 1 2 0 0 1 0 0

carbon dioxide + 2-Acetolacetate 2 Pyruvate + H+

CO2 + C5H7O4- 2 C3H3O3

- + H+

Merge

• Works on simple premises

• Metabolites / enzymes are the same if the share the same annotation and compartment

• Reactions are the same if the have the same stoichiometry• Hence balancing• Isoenzymes collated (“OR” relationship in GPR

definition)

“Fuzzy” Merge

• Sources can be inconsistent in their definition of stereochemical precision

• Considered different: apparently minor but can cause gaps in the network

beta-D-glucose D-glucose

“Fuzzy” Merge

• ChEBI is an ontology and contains relationships between metabolites• Edges not just nodes

Compartment

• Determination of intracellular compartment in which enzymes operate

• Two approaches:• Extract curated information from UniProt annotation• Extract protein sequence from UniProt and pipe to

WoLF PSORT localisation prediction algorithm

• Infer reaction localisation from enzyme localisation• Hypothetical transport reactions added to be

validated / deleted in manual curation phase

Transporters and biomass

• Extracellular and intracellular transporters are required

• TransportDB and Transporter Classification DB are sources of transporter proteins• BUT not comprehensive enough to automatically

assign these to individual reactions

• Biomass reaction should be added manually

• Automation breaks down…

Shorthand

Converted to SBML, protonated, balanced and merged as before

Usage and availability

• Java programming API

• Simple command line interface• SBML file in / SBML file out

• Has some dependencies on third-party software• Free to academic users

• http://www.mcisb.org/subliminal/

Thanks…

The Subliminal Toolbox: automating steps in the

reconstruction of metabolic networks

Neil SwainstonManchester Centre for Integrative Systems Biology

1st Conference on Constraint-based Reconstruction and AnalysisReykjavik, Iceland

24 June 2011

UniProt compartmentalisation

UniProt compartmentalisation

UniProt curated information

PSORT compartmentalisation

PSORT compartmentalisation

WoLF PSORT prediction