Transcript Reconstruction and comparative analysis of the metabolic network

Metabolic peculiarities of
Aspergillus niger disclosed by
comparative metabolic genomics
Authors : Jibin Sun1 , Xin Lu1 , Ursula Rinas1 and
An Ping Zeng1,2
1Helmholtz
2Hamburg
Centre for Infection Research, Inhoffenstr., 38124 Braunschweig, Germany
University of Technology, Institute of Bioprocess and Biosystems Engineering,
Denickestr., 21071 Hamburg, Germany
Presented By -> Gaurav Jain
{ [email protected] }
Instructor : Dr. Li Liao
{[email protected] }
1
In Short
• A.niger : an important industrial microorganism.
• Used for the production of :
–
metabolites : such as citric acid.
–
Proteins: such as fungal enzymes or heterologous proteins.
Despite its extensive industrial applications, the genetic
inventory of this fungus is only partially understood. The
recently released genome sequence opens a new horizon for
both scientific studies and biotechnological applications.
2
Overview
• Introduction
– Terminologies
– Background
• Results and Discussion
– Genomic annotation of ATCC 9029
– Comparative genomics
• Reconstruction and comparative analysis of the metabolic network
– Metabolic network reconstruction
– Comparative assessment of the central metabolic network
– Unique enzyme-coding genes and EC numbers
• Citric acid production: A case study
– Versatile metabolic conversion centre
• My opinion
• Conclusion
3
Terminologies
•
Metabolism: The chemical processes occurring within a living cell or organism that
are necessary for the maintenance of life. In metabolism some substances are
broken down to yield energy for vital processes while other substances, necessary
for life, are synthesized.
•
Metabolite : Organic compound that is a starting material in, an intermediate in,
or an end product of metabolism.
•
Ortholog or orthologous genes: are genes in different species, that are similar to
each other because they originated from a common ancestor. The term
"ortholog" was coined in 1970.
•
CD`s : Coding sequences, of a gene is the portion of DNA that is transcribed into
mRNA and translated into proteins. This does not include such regions as a
recognition site, initiator sequence, or termination sequence, only the region
that will directly code for amino acid linkage.
•
Enzyme Commission number (EC number) is a numerical classification scheme for
enzymes, based on the chemical reactions they catalyze. Strictly speaking, EC
numbers do not specify enzymes, but enzyme-catalyzed reactions. If different
enzymes (for instance from different organisms) catalyze the same reaction, then
they receive the same EC number.
4
Background
• Importance of metabolic network reconstruction.
• Modern fast DNA sequencing methods & bioinformatics tools
for reconstruction and cross comparison of the metabolic
networks among related species and specific strains-elucidate
metabolic peculiarities.
• Why there is a great need for a better knowledge of genomic
potential of A.niger?
– Well known producer of extracellular fungal enzymes.
– Citric acid is mostly exclusively produce d using A.niger nowadays.
– Could be used for rational strain improvement.
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Background Contd…
• Reconstruction of a genome scale metabolic network from
the annotated genome:
– List of enzymes with EC numbers are extracted from the genome
annotation.
– It is then searched in an established biochemical reaction DB to
acquire their corresponding reactions.
– Biochemical reactions are then connected to each other according to
certain rules.
– Such information can be further interpreted as a network and
analyzed by many computer programs.
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Next…
• Introduction
– Terminologies
– Background
• Results and Discussion
– Genomic annotation of ATCC 9029
– Comparative genomics
• Reconstruction and comparative analysis of the metabolic network
– Metabolic network reconstruction
– Comparative assessment of the central metabolic network
– Unique enzyme-coding genes and EC numbers
• Citric acid production: A case study
– Versatile metabolic conversion centre
• My opinion
• Conclusion
7
Genomic annotation of ATCC
9029
• Annotated raw genome (approx 32Mb) sequence of A.niger
ATCC 9029 from Integrated genomics was annotated by using
a new algorithm ‘IdentiCS’ with a cutoff E-value of 10-5 .
• ‘IdentiCS’ is a homology-based algorithm.
• The algorithm was extended for the prediction and
annotation of eukaryotic CDSs by considering the intron and
extron structure of genes.
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GenomiC annotation of atCC 9029 Contd…
• Complementation of ‘IdentiCS’ by GenScan and GeneWise for the
prediction of protein-encoding genes.
– Like all other homology-based methods, ‘IdentiCS’ Is unable to predict
new genes for which no homologue is present in the available protein
database.
• Refinement of the annotation
1.
All CDSs predicted submitted to KEGG automatic Annotation Server.
2.
Use ‘HT-Go-FAT ‘ useful software toolkit to annotate sequences to GO, EC
number, KEGG pathways and so on.
3.
Use of text mining to assign EC number when an obvious enzyme could
not be associated to an EC number or a complete EC number using above
methods.
9
Functional annotation of predicted protein coding sequences of A. niger.
Sun et al. Genome Biology 2007 8:R182 doi:10.1186/gb-2007-8-9-r182
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Comparative Genomics
• Proteins predicted from the unfinished genomic sequences of
A.niger ATCC 9029 and from A.niger CBS 513.88 were cross
compared with proteins of 7 fungal species and 26 representative
from eukaryotic organism to identify their orthologus relationships.
• Results :
– 88% of the CDSs can be unique to a fungus in comparison to another fungal
species.
– Nearly 50% of the CDSs of A. niger CBS 513.88 cannot be found in other
Aspergillus species
– Over 30% of the CDSs in each A. niger strain have homologs in another A.
niger strain with an identity level higher than 99%.
– The unique genes account for around 4% of the total number of CDSs in the
two A. niger strains.
In summary, the results from comparative genomics show that the A. niger strains
are closely related to each other but exhibit large differences from the other fungal
species compared.
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Next…
•
Introduction
– Terminologies
– Background
•
Results and Discussion
– Genomic annotation of ATCC 9029
– Comparative genomics
• Reconstruction and comparative analysis of the metabolic network
– Metabolic network reconstruction
– Comparative assessment of the central metabolic network
– Unique enzyme-coding genes and EC numbers
•
Citric acid production: A case study
– Versatile metabolic conversion centre
•
•
My opinion
Conclusion
12
Section Overview
•
•
•
•
Metabolic network reconstruction
Quick tour of KEGG
Using KEGG to reconstruct a metabolic network
Reconstruction of a simple pathway
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Metabolic network reconstruction
• Identification of missing genes in complete genomes
• Search for candidates
– Analysis of individual genes to assign general
biochemical function:
• homology
• functional patterns
• structural features
– Comparative genomics to predict specificity:
•
•
•
•
analysis of regulation
positional clustering
gene fusions
phylogenetic patterns
14
metaboliC network reConstruCtion Contd…
15
Tour of KEGG
• KEGG: Kyoto Encyclopedia of Genes and Genomes.
• Main entry points to the KEGG web service
– KEGG2 KEGG Table of Contents
– PATHWAYKEGG pathway maps for biological processes
– BRITE Functional hierarchies of biological systems
– GENES Gene catalogs and ortholog relations in complete genomes
– LIGAND Chemical compounds, drugs, glycans, and reactions
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Information about A.niger
17
KEGG pathway database
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Making an Organism Specific map
Enzymes
in
A.niger
are
now
shaded in
green
19
Fungus-specific information about
EC 2.7.1.1
ORF , Gene name
Reaction
Information
Databases
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Reaction
Information
21
Enzyme Database
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First Entry in the reconstruction
• ORF : An02g14380
• GENE : hxk
• Name: ATP:D-hexose 6phosphotransferase
• EC number : 2.7.1.1
• Reaction
An02g14380
hxk
HXK1
ATP + D-hexose = ADP + D-hexose 6-phosphate
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Metabolic network reconstruction of
A.niger
• The metabolic network of A. niger was constructed using the EC
numbers of ATCC 9029 and CBS 513.88 strains.
• Methods applied:
– knowledge-based method.
– connection-matrix-based method.
• Results:
– 2443 biological reactions and 2349 metabolites.
– Most of the reactions are connected to central metabolisms like
carbohydrate, lipid, amino acid, energy metabolism etc.
– a significant number of reactions and metabolites belong to secondary
metabolism or xenobiotics biodegradation, indicating the high metabolic
potential of A. niger for production of secondary metabolites or for
bioremediation
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The genome-scale metabolic network of A. niger. Nodes are metabolites while links are reactions. The
color of the nodes represents different functional categories. The size of nodes is proportional to the
number of reactions from or to that node (metabolite) in the genome-wide network. (a) The general layout
of the metabolic network. (b) A zoom-in of the dashed box in
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A comparative assessment of the central
metabolic network
• The metabolic network reconstructed from the genomic data was
compared to the network of central carbon metabolism of A. niger
reconstructed by David et al.
• The network of David et al. contained 335 reactions, 284
metabolites and 129 EC numbers.
• In general, there is a good agreement between these two metabolic
networks regarding central metabolism.
• Only 14 ECs in the metabolic network of David et al. could not be
found in the genome-wide network reconstructed, most of which
belong to enzymes poorly characterized in the literature in terms of
protein sequences.
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Uniqueness of genes or CDSs
• The uniqueness of a gene or CDS from organism A is defined as :
If the Ortholog of a gene from organism A is absent in organism B, it is
defined that this gene is unique or specific to organism A in
comparison to organism B.
• This does not imply there is no homolog (namely paralog) of the
gene from organism A in organism B. In some cases, this gene is
just an additional copy of another gene whose orthologs are
found in both organisms.
• This also does not imply that this gene is found only in organism
A.
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Unique enzyme-coding genes
and EC numbers
Glycolysis and TCA cycle of A. niger: a view from the genomescale network. Nodes represent metabolites while directional links
represent metabolic reactions. The color of the nodes represents
different functional categories. The size of nodes is proportional to
the number of reactions from or to that node (metabolite) in the
genome-wide network. The red colored links indicate that A. niger
has additional copies of genes for these reactions
• The unique enzyme-coding genes mentioned
do have paralogs in other fungi or even in A.
niger itself.
• These paralogs were carefully verified not to be
orthologs since they are orthologous to other
CDSs of A. niger.
• Gene redundancy or duplication has also been
reported in A. niger
28
Next…
• Introduction
– Terminologies
– Background
• Results and Discussion
– Genomic annotation of ATCC 9029
– Comparative genomics
• Reconstruction and comparative analysis of the metabolic network
– Metabolic network reconstruction
– Comparative assessment of the central metabolic network
– Unique enzyme-coding genes and EC numbers
• Citric acid production: A case study
– Versatile metabolic conversion centre
• My opinion
• Conclusion
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A versatile metabolic conversion
center
• Importance of citric acid production by A. niger, the metabolic
reactions contributing to citric acid production are selected as an
example to explore the capability of the constructed metabolic
network.
• The pathways related to citric acid production from glucose were
extracted from the genome-wide metabolic network together with
the metabolites directly connected to these pathways.
• In comparison to other filamentous fungi, A. niger has redundant
unique genes for the conversion of seven of (starch, sucrose, dextrin,
maltose, lactose, cellulose, α,α-trehalose, sorbitol, D-glucoside, N-glycan)
substrates (reactions marked as red in Figure).
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Next…
• Introduction
– Terminologies
– Background
• Results and Discussion
– Genomic annotation of ATCC 9029
– Comparative genomics
• Reconstruction and comparative analysis of the metabolic network
– Metabolic network reconstruction
– Comparative assessment of the central metabolic network
– Unique enzyme-coding genes and EC numbers
• Citric acid production: A case study
– Versatile metabolic conversion centre
• My opinion
• Conclusion
31
My opinion
• I find the paper was good as it looked certain metabolic
peculiarities of
A.niger which is an important industrial
microorganism.
• As they were first to present the genome-scale metabolic network
for A. niger and an in-depth genomic comparison of this species, I
opened a huge space in front of the potential of A.niger.
• The paper was not organized properly.
• Many things which were said to be mentioned in the “materials and
methods" sectioned was not proper.
• Lot of data which they could have represented by tables were given
as additional data.
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Conclusion
•
14,000 protein coding sequences were predicted from the raw low-coverage
genome sequence of A. niger ATCC 9029 and approximately 60% of them were
assigned to at least one functional category (GO, KO, COG, EC and pathways).
•
It is found that the genomic content of A. niger ATCC 9029 is very similar to
that of A. niger CBS 513.88; merely around 600 genes are exclusively found in
each strain.
•
Comparative metabolic genomics revealed the high metabolic peculiarity of A.
niger by more than 1,100 unique enzyme-encoding genes.
•
Many unique genes are paralogsof those genes that are orthologs in the
compared fungi, indicating that genetic multiplicity might be a key strategy of
A. niger to keep its versatile metabolic capacities and its robustness to adapt
to different environmental conditions.
•
Additional copies of genes, such as the ones encoding alternative
mitochondrial oxidoreductase and citrate synthases, which could have an
impact on the overproduction of citric acid by this black mould.
33
References
•
Data Collection and Metabolic Network Reconstruction -Winter
School in Genomics, 2006.
•
Metabolism of Citric Acid Production by Aspergillus niger :
Model
Definition,
Steady-State
Analysis
and
Constrained
Optimization of Citric Acid Production Rate by Fernando
Alvarez-Vasquez,1 Carlos Gonza´ lez-Alco´ n,2 Ne´ stor V.
Torres1.
•
Metabolic peculiarities of Aspergillus niger disclosed by
comparative metabolic genomics Authors : Jibin Sun , Xin Lu ,
Ursula Rinas and An Ping Zeng
•
http://www.genome.jp/kegg/
•
http://www.ncbi.nlm.nih.gov/
•
http://www.google.com/
•
Help of Dr.Liao.
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Questions
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