Download The construction of LacplantCyc and the use of physiological and

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
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(2003) Proc Natl Acad Sci USA 100,1990
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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