Reconstructing the metabolic network of a bacterium from its genome: the construction of LacplantCyc

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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
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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
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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)
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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
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We are interested in lactic acid bacteria

(2003) Proc Natl Acad Sci USA 100,1990
annotation database PlantDB
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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
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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
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the actual labour curation
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What we have done consult reference databases
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What we have done add information that is not
recovered from MetaCyc.
Transporters are not recovered by pathway tools
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What we have done add information that is not
recovered from MetaCyc.
pyruvate
PEP
- include newly discovered and or organism
specific reactions and pathways - add information
on complex formation
EI
EI
P
HPr
HPr
P
170
170
P
dak1
dak2
dak1
170
dak2
P
dihydroxy acetone
dihydroxy acetone
P
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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
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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
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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
passive
experimental maltose phosphorylase
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the evaluation of gaps in pathways are
genes really missing
?
?
an example Tetrahydrofolate synthesis by
Lactobacillus plantarum
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the evaluation of gaps in pathways
track missing genes
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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
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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
gt In all reactions succinyl-CoA is used as a
substrate it has to be replaced by acetyl-CoA
TCA cycle
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cleaning up the database
the removal of redundant pathways
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a comparison of the automatic and curated
LacplantCyc
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using LacplantCyc
- inconsistencies between observed nutrient
requirements and pathway predictions may lead to
new insights about regulation
automatic
?
further research needed
?
?
?
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using LacplantCyc
- to visualize -omics data - to compare the
metabolic network between different species
we need improved visualization
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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
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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
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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
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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.
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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