Reconstructing the metabolic network of a bacterium from its genome: the construction of LacplantCyc Christof Francke In silico reconstruction of the metabolic pathways of Lactobacillus plantarum: comparing predictions of nutrient requirements with growth experiments Te usink, van Enckevort, Francke, Wiersma, Wegkamp, Smid and Siezen 2005 Appl. Environ. Microbiol. 71:

What do we mean by reconstruction? the collection and visualization of all potential physiologically relevant cellular processes Reconstructing the metabolic network of a bacterium from its genome Francke, Siezen and TeusinkTrends in Microbiol :

Why do we want to do it? it serves to sort the individual proteins and thus the potential molecular functions, into a context (like pathways or protein complexes) and as such: - allows for improved functional annotation - provides a platform to visualize and analyze 'omics' data - yields a network the topology of which can be studied - can be converted to a model (metabolic engineering)

How do we annotate? The attribute function is ambiguous context independent (molecular function or properties) - catalyze certain reactions - interact with certain proteins - bind to a specific DNA sequence context dependent (role) - act in a certain pathway - be a member of a certain protein complex(es) - act as a transcription factor

We are interested in lactic acid bacteria (2003) Proc Natl Acad Sci USA 100,1990 #### annotation database PlantDB

recovering gene - protein - reaction -pathway relations the construction of LacplantCyc Pathway Tools (SRI) uses gene-annotation (EC-numbers) and reference database (MetaCyc) to arrive automatically at an encyclopedia of genes connected to proteins proteins connected to reactions reactions connected in pathways

initial automatic reconstruction: some remarks - presence of pathways - gaps in pathways - same reactions and pathways Are the assignments correct and which functions are there that have not been retrieved? - are these numbers correct? - manual changes are not correctly incorporated

the actual labour: curation

What we have done: consult reference databases

What we have done: add information that is not recovered from MetaCyc. Transporters are not recovered by pathway tools

~P EI PEP pyruvate ~P HPr ~P 170 dak1 dak2 dak1 dak2 ~P 170 dihydroxy acetone ~P - include newly discovered and or organism specific reactions and pathways - add information on complex formation What we have done: add information that is not recovered from MetaCyc.

What we have done: evaluation of the attributed molecular function for each individual case Do we trust the gene - protein - reaction association when we consider the similarity between the sequence of the gene-product and the sequence of a protein with the specified molecular function (evidence based on experiment)? - determine orthology (use phylogeny and gene-context to determine evolutionary relationship) - check experimental evidence

The evaluation of the attributed molecular function: Improved annotation of homologous proteins the use of phylogeny and orthologous relations experimental: trehalose phosphorylase map4 experimental: kojibiose phosphorylase map1* * there are slight but significant differences in alignment of cluster 1 which might point to slightly altered specificity L. plantarum has four homologs annotated as maltose phosphorylase

The evaluation of the attributed molecular function: Improved annotation of homologous proteins the use of gene context and metabolic context map3 experimental: maltose phosphorylase map2 experimental: maltose phosphorylase activepassive

the evaluation of gaps in pathways: are genes really missing an example: Tetrahydrofolate synthesis by Lactobacillus plantarum  ?

the evaluation of gaps in pathways: track missing genes

the evaluation of gaps in pathways: the use of knowledge on physiology Validation: no tetrahydrofolate detectable without addition of p-aminobenzoate to the medium absent predicted growth dependence

the evaluation of reactions and pathways: the use knowledge on physiology TCA cycle Lactobacilli do not have a TCA cycle and therefore do not produce succinyl-CoA ==> In all reactions succinyl-CoA is used as a substrate it has to be replaced by acetyl-CoA

cleaning up the database the removal of redundant pathways

a comparison of the automatic and curated LacplantCyc

using LacplantCyc - inconsistencies between observed nutrient requirements and pathway predictions may lead to new insights about regulation further research needed  automatic   

using LacplantCyc - to visualize -omics data - to compare the metabolic network between different species we need improved visualization

About the use of Cyc to visualize 'omics' data - it preferably requires * an interactive overview with more information * the possibility of having multiple selectable overviews * colouring of the genes instead of the reactions remarks

using LacplantCyc - to help the reconstruction of the metabolic network of a related species through orthologous relationships between proteins - to serve as the starting point for making a metabolic model (constraint based modeling) Accelerating the reconstruction of genome-scale metabolic networks Notebaart, van Enckevort, Francke, Siezen and Teusink in preparation

About the use of: Cyc as a source of gene-reaction-pathway association information to be used in other applications - requires easy export of these associations Cyc as a starting point for modeling - requires balanced reactions, detailed and correct molecular information on compounds and balance checks remarks

Pathway Tools is very nice to quickly connect reaction and pathway information to a gene which has been annotated with an EC-code. However: - Generation of a reliable reconstruction requires a lot of work and the implementation of changes is not always straightforward (problems with certain frames) and requires a lot of steps. - Better control over the editor of individual pathways and the pathway overview would be an important asset. - Application of automatic procedures after curation unfortunately destroys the changes that were carefully implemented. - Multiple editors with straightforward import and export functions would enhance the usefulness. final remarks

acknowledgements Frank van Enckevort Christof Francke Richard Notebaart Roland Siezen Eddy Smid Bas Teusink Arno Wegkamp Anne Wiersma LacplantCyc can be found at