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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

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Christof Francke


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slide1

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

slide2

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

slide3

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)

slide4

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

slide5

We are interested in lactic acid bacteria

#

#

(2003) Proc Natl Acad Sci USA 100,1990

annotation database

PlantDB

slide6

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

slide7

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

slide9

What we have done:

consult reference databases

slide10

What we have done:

add information that is not recovered from MetaCyc.

Transporters are not recovered by pathway tools

slide11

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

slide12

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 thesimilarity 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

slide13

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

slide14

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

slide15

the evaluation of gaps in pathways:

are genes really missing

?

an example: Tetrahydrofolate synthesis by Lactobacillus plantarum

slide17

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

slide18

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

slide19

cleaning up the database

the removal of redundant pathways

slide21

using LacplantCyc

- inconsistencies between observed nutrient requirements

and pathway predictions may lead to new insights about regulation

automatic

further research needed

slide22

using LacplantCyc

- to visualize -omics data

- to compare the metabolic network between different species

we need improved visualization

slide23

remarks

About the use ofCyc 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

slide24

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

slide25

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

slide26

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.

slide27

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