metabolic peculiarities of aspergillus niger disclosed by comparative metabolic genomics

Metabolic peculiarities of Aspergillus niger disclosed by

comparative metabolic genomicsAuthors : Jibin Sun1 , Xin Lu1 , Ursula Rinas1 and

An Ping Zeng1,2 1Helmholtz Centre for Infection Research, Inhoffenstr., 38124 Braunschweig, Germany

2Hamburg 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] }

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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.

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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

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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.

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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






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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 annotation1. 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.

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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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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

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Metabolic network reconstruction Contd…

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Tour of KEGG

• KEGG: Kyoto Encyclopedia of Genes and Genomes.

• Main entry points to the KEGG web service– KEGG2 KEGG Table of Contents

– PATHWAYKEGG 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

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KEGG pathway database

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Making an Organism Specific map

Enzymes in

A.niger are now

shaded ingreen

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Fungus-specific information about

EC 2.7.1.1

ORF , Gene name

Reaction Information

Databases

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Reaction Information

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Enzyme Database

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First Entry in the reconstruction

• ORF : An02g14380• GENE : hxk• Name: ATP:D-hexose 6-

phosphotransferase • EC number : 2.7.1.1• Reaction

An02g14380

hxk

ATP + D-hexose = ADP + D-hexose 6-phosphate

HXK1

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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 numbersGlycolysis and TCA cycle of A. niger: a view from the genome-scale 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

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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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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.

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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

Metabolic

peculiaritie

s ???

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metabolic peculiarities of aspergillus niger disclosed by comparative metabolic genomics

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