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Reconstructing the metabolic network of a bacterium from its genome: the construction of LacplantCyc In silico reconstruction of the metabolic pathways of Lactobacillus plantarum: comparing predictions of nutrient requirements with growth experiments Teusink, van Enckevort, Francke, Wiersma, Wegkamp, Smid and Siezen 2005 Appl. Environ. Microbiol. 71: 7253-7262 Christof Francke 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 Teusink Trends in Microbiol. 2005 13: 550-558 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 the construction of LacplantCyc recovering gene - protein - reaction -pathway relations 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 Are the assignments correct and which functions are there that have not been retrieved? - presence of pathways - gaps in pathways - are these numbers correct? - same reactions and pathways - 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 What we have done: add information that is not recovered from MetaCyc. - include newly discovered and or organism specific reactions and pathways - add information on complex formation pyruvate EI PEP EI ~P HPr ~P HPr 170 170 ~P dak1 170 dak2 ~P dihydroxy acetone dak1 dak2 dihydroxy acetone ~P 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: kojibiose phosphorylase map1* experimental: trehalose phosphorylase map4 * 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 map2 map3 experimental: maltose phosphorylase active experimental: maltose phosphorylase passive the evaluation of gaps in pathways: A B are genes really missing ? an example: Tetrahydrofolate synthesis by Lactobacillus plantarum the evaluation of gaps in pathways: track missing genes C D Gene identifier product name gene name EC-code lp_0014 lp_0119 lp_0897 lp_0962 lp_1144 lp_1627 lp_1737 lp_1885 replicative DNA helicase DnaC NTP pyrophosphohydrolase NTP pyrophosphohydrolase (putative) ATP-dependent DNA helicase RecQ ATP-dependent DNA helicase PcrA ATP-dependent DNA helicase RecG ATP-dependent helicase DinG ATP-dependent DNA helicase RecQ dnaC lp_0119 lp_0897 recQ1 pcrA recG dinG recQ2 3.6.1.3.6.1.3.6.1.3.6.1.3.6.1.3.6.1.3.6.1.3.6.1.- Lp_3295 xanthosine triphosphate pyrophosphatase xtp2 no EC code the evaluation of gaps in pathways: the use of knowledge on physiology absent absent predicted growth dependence Validation: no tetrahydrofolate detectable without addition of p-aminobenzoate to the medium the evaluation of reactions and pathways: the use knowledge on physiology 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 TCA cycle cleaning up the database the removal of redundant pathways a comparison of the automatic and curated LacplantCyc - inconsistencies between observed nutrient requirements and pathway predictions may lead to new insights about regulation automatic further research needed using LacplantCyc using LacplantCyc - to visualize -omics data - to compare the metabolic network between different species we need improved visualization remarks 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 using LacplantCyc - to help the reconstruction of the metabolic network of a related species through orthologous relationships between proteins Accelerating the reconstruction of genome-scale metabolic networks Notebaart, van Enckevort, Francke, Siezen and Teusink - to serve as the starting point for making a metabolic model (constraint based modeling) in preparation remarks 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 final 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. acknowledgements Frank van Enckevort Christof Francke Richard Notebaart Roland Siezen Eddy Smid Bas Teusink Arno Wegkamp Anne Wiersma LacplantCyc can be found at www.lacplantcyc.nl