Metabolic Network Analysis Algorithms in Pathway · PDF fileAlgorithms in Pathway Tools Peter D. Karp, Ph.D. Bioinformatics Research Group. ... Inference tools z Analyzing biological

1 SRI International Bioinformatics

Metabolic Network Analysis Algorithms in Pathway Tools

Peter D. Karp, Ph.D.Bioinformatics Research Group

SRI [email protected]

BioCyc.orgEcoCyc.org, MetaCyc.org, HumanCyc.org


Systems Biology

Def 1: System-scale descriptions and analyses of biological sytems

Def 2: Predictive modeling of biological systems


Overview

Pathway/Genome DatabasesBioCyc collectionEcoCyc, MetaCyc

Pathway Tools softwareVisualization, Editing, AnalysisInference tools

Analyzing biological networks to identify gaps and inconsistenciesPrediction of growth media from metabolic network


What to do When Theories BecomeLarger than Minds can Grasp?

Example: E. coli metabolic network244 pathways involving 1,029 reactions and 895 substrates

Example: E. coli genetic networkControl by 97 transcription factors of 1174 genes in 630 transcription units

Past solutions:Experts specializePublish theories in textual form

We cannot compute with theories in those formsEvaluate theories for consistency with new data: microarraysRefine theories with respect to new data Compare theories describing different organisms


Databases of Metabolic Pathway Data

Organize growing corpus of data on metabolic pathwaysExperimentally elucidated pathways in the biomedical literatureComputationally predicted pathways derived from genome data

Provide software tools for querying and comprehending this complex information space

Multiorganism view: MetaCycUnique, experimentally elucidated pathways across all organismsReference database for computational pathway prediction

Organism-specific view:Organism-specific Pathway/Genome DatabasesDetailed qualitative models of metabolic networks Combine computational predictions with experimentally determined pathways


Pathway Tools Capabilities

Create and maintain an organism database integrating genome, pathway, regulatory information

Computational inference toolsInteractive editing tools

Query and visualize that databaseUse the database to interpret omics dataMetabolic network analysis toolsComparative analysis tools


BioCyc Collection of 507 Pathway/Genome Databases

Pathway/Genome Database (PGDB) –combines information about

Pathways, reactions, substratesEnzymes, transportersGenes, repliconsTranscription factors/sites, promoters, operons

Tier 1: Literature-Derived PGDBsMetaCycEcoCyc -- Escherichia coli K-12

Tier 2: Computationally-derived DBs, Some Curation -- 24 PGDBs

HumanCycMycobacterium tuberculosis

Tier 3: Computationally-derived DBs, No Curation -- 481 DBs


Pathway Tools Software

PathoLogicPredicts operons, metabolic network, pathway hole fillers, from genomeComputational creation of new Pathway/Genome Databases

Pathway/Genome EditorsDistributed curation of PGDBsDistributed object database system, interactive editing tools

Pathway/Genome NavigatorWWW publishing of PGDBsQuerying, visualization of pathways, chromosomes, operonsAnalysis operations

Pathway visualization of gene-expression dataGlobal comparisons of metabolic networks

Bioinformatics 18:S225 2002


EcoCyc Project – EcoCyc.orgE. coli Encyclopedia

Review-level Model-Organism Database for E. coliTracks evolving annotation of the E. coli genome and cellular networksThe two paradigms of EcoCyc

“Multi-dimensional annotation of the E. coli K-12 genome”Positions of genes; functions of gene products – 76% / 66% expGene Ontology terms; MultiFun termsGene product summaries and literature citationsEvidence codesMultimeric complexesMetabolic pathwaysCellular regulation

Nuc. Acids Res. 35:7577 2007 ASM News 70:25 2004 Science 293:2040

Karp, Gunsalus, Collado-Vides, Paulsen


EcoCyc = E.coli Dataset + Pathway/Genome Navigator

Genes: 4,478

Proteins: 4,479Complexes: 880

RNAs: 285

Reactions:Metabolic: 975Transport: 272

Pathways: 237

Compounds: 1,373

URL: EcoCyc.org

Gene Regulation:Operons: 3,359Trans Factors: 196Promoters: 1,766

TF Binding Sites: 2,105

EcoCyc v13.5

Citations: 19,000


Paradigm 1:EcoCyc as Textual Review Article

All gene products for which experimental literature exists are curated with a minireview summary

Found on protein and RNA pages, not gene pages!3257 gene products contain summaries

Summaries cover function, interactions, mutant phenotypes, crystal structures, regulation, and more

Additional summaries found in pages for operons, pathways

EcoCyc cites 17,300 publications


Paradigm 2: EcoCyc as Computational Symbolic Theory

Highly structured, high-fidelity knowledge representation provides computable informationEach molecular species defined as a DB object

Genes, proteins, small moleculesEach molecular interaction defined as a DB object

Metabolic reactionsTransport reactionsTranscriptional regulation of gene expression

220 database fields capture extensive properties and relationships


EcoCyc Accelerates Science

ExperimentalistsE. coli experimentalistsExperimentalists working with other microbesAnalysis of expression data

Computational biologists Biological research using computational methodsGenome annotationStudy connectivity of E. coli metabolic networkStudy phylogentic extent of metabolic pathways and enzymes in all domains of life

BioinformaticistsTraining and validation of new bioinformatics algorithms – predict operons, promoters, protein functional linkages, protein-protein interactions,

Metabolic engineers“Design of organisms for the production of organic acids, amino acids, ethanol, hydrogen, and solvents “

Educators


MetaCyc: Metabolic Encyclopedia

Describe a representative sample of every experimentally determined metabolic pathwayDescribe properties of metabolic enzymes

Literature-based DB with extensive references and commentaryPathways, reactions, enzymes, substrates

Jointly developed by P. Karp, R. Caspi, C. Fulcher, SRI InternationalL. Mueller, A. Pujar, Cornell UnivS. Rhee, P. Zhang, Carnegie Institution

Nucleic Acids Research 2008


Applications of MetaCyc

Reference source on metabolic pathways

Metabolic engineeringFind enzymes with desired activities, regulatory propertiesDetermine cofactor requirements

Predict pathways from genomes

Systematic studies of metabolism

Computer-aided education


MetaCyc Data -- Version 13.5

Pathways 1,400

Reactions 8,100

Enzymes 5,900

Small Molecules 8,200

Organisms 1,800

Citations 20,800


Taxonomic Distribution ofMetaCyc Pathways – version 13.1

Bacteria 883

Green Plants 607

Fungi 199

Mammals 159

Archaea 112


Pathway Tools Overviews and Omics Viewers

Designed to avoid the hairball effectGenerated automatically from PGDBMagnify, interrogateOmics viewers paint omics data onto

overview diagramsDifferent perspectives on same datasetUse animation for multiple time points or conditionsPaint any data that associates numbers with genes, proteins, reactions, or metabolites

Genome-scale visualizations of cellular networksHarness human visual system to interpret patterns in biological

contexts


Regulatory Overview and Omics Viewer

Show regulatory relationships among gene groups



Genome Poster


Dead End MetabolitesClues to extra/missing reactions A small molecule C is a dead-end if:

(Def 1 easier to compute; Def 2 more accurate)Definition 1:

C is a substrate in only one reaction of the set of SMM reactions occurring in Compartment ANDNo reactions exist containing parent classes of C ANDNo transporter acts on C in Compartment, nor on parent classes of C

Definition 2:C is produced only by SMM reactions in Compartment, and no transporter acts on C in Compartment ORC is consumed only by SMM reactions in Compartment, and no transporter acts on C in Compartment


Dead-End Metabolite Analysis of E. coli

36 22 dead ends in metabolic pathways174 dead ends in full metabolic network

GDP-L-fucoseProduced onlyLiterature research supported addition of a reaction producing colanic acid from GDP-L-fucose

D-galactarate and D-glucarateDegraded onlyLiterature indicates both can be used as C sourcesHypothetical transport reactions addedProbable gene identified through knock-out study


Reachability Analysis of Metabolic Networks

Given:A PGDB for an organismA set of initial metabolites

Infer:What set of products can be synthesized by the small-molecule metabolism of the organism

Motivations:Quality control for PGDBsVerify that a known E. coli growth medium yields known essential compounds of E. coliRomero and Karp, Pacific Symposium on Biocomputing, 2001


Algorithm: Forward PropagationThrough Production System

Each reaction becomes a production ruleEach of the 21 metabolites in the nutrient set becomes an axiom

Nutrientset

Metabolitepool

“Fire”reactions

Products

Reactants

PGDBreaction

set

A + B C



Results from EcoCyc Reachability Analysis in 2001

Phase I: Forward propagation21 initial compounds yielded only half of the 41 essential compounds for E. coli

Phase II: Manually identifyBugs in EcoCyc (e.g., two objects for tryptophan)

A B B’ CIncomplete knowledge of E. coli metabolic network

A + B C + D“Bootstrap compounds”Missing initial protein substrates (e.g., ACP)

Protein synthesis not represented

Phase III: Forward propagation with 11 more initial metabolites

Yielded all 41 essential compounds


Minimal Nutrient Sets

Carolyn Talcott, Markus Krummenacker, Steven Eker, and Peter Karp

Computer Science Laboratory and

Bioinformatics Research Group

SRI, InternationalOctober 21st, 2009


The Problem

Given a model of metabolism for an organism, determine minimal sets of nutrients that will support growth.

Model -- network of metabolic reactions (R)Nutrients -- transportables (T), compounds that have transport reactionsGrowth -- production of essential compounds (E)

A subset N of T is a nutrient set if E is R-producible from NN is minimal if no proper subset is a nutrient set


Mathematical Approach

S = stochiometric matrix for R Sij coeff of Ci in Rj

r = vector of reaction fluxesp = S x r -- production pi is production rate of Ci

pi = Si1 r1 + .... + Sik rk

Basic constraintsri >= 0 -- reactions run forwardpi > 0 if Ci in Epi >= 0 if Ci not in E or NIf a compound Cj not in E or T is used, it must be

produced (pj > 0)


Problem Simplification

Impossibility eliminationDrop reactions that have reactants that can not be produced (or transported)(Uses forward collection)

Uselessness eliminationDrop useless compounds and reactions whose products are all uselessThe useful compounds are found by backwards propagation from E (Uses backwards collection) �


Searching for Minimal Nutrient Sets

Define nutset(N) for N a subset of T bynutset(N) = true if the constraints for N are satisfiable

= false otherwiseUse a constraint solver (Yices) to determine if there is a solutionFind one minimal N: Start with N = T and eliminate elements until no more can be eliminated.Finding all requires some cleverness to do it feasibly. Our approach uses a representation of Boolean functions called BDDs (binary decision diagrams) to search for extensions of a set of minimal solutions. �


E. coli Case Study

160 Transportables1378 Compounds2251 Reactions36 Essentials

1156 Solutions9 Reduced solutions


Some Minimal Nutrient Sets

Solution 5TaurinePhosphateL-alanine

Solution 6TaurinePhosphateL-aspartate


Equivalence and Reduced Solutions

Problem: Large number of minimal nutrient sets (1156) is hard to understand and evaluateSolution: Nutrient equivalence classes

Define two nutrients A,B to be equivalent if whenever A appears in a minimal nutrient set, then replacing A by B yields another minimal nutrient set, and conversely

Benefits: Small number of solutionsInsights into the role of each nutrient


One Reduced Solution and its Equivalence Classes

Reduced solution 5CytidineSulfatePhosphate

Equivalence Classes:(CN): cytidine, 32 other compounds, L-alanine, L-aspartate(S): taurine, sulfate(P): phosphate


Lessons Learned

Analysis is a great way to debug a knowledge baseGaps in network Missing participantsIncorrect reaction directions


Ten Equivalence Classes

2 Unitary:HPO4 (P)nicotinamide mononucleotide (CNP)

3 with two compounds:Sulfate / taurine (S) L-methionine / glutathione (CNS)beta-D-glucose-6-phosphate / sn-glycerol-3-phosphate (CP)

1 Medium (9 cpds)L-valine/NH4+/ … (N)

2 Very largefumarate/malate/ ... (C) -- 50 cpdscytidine/L-aspartate/ ... (CN) – 35 cpds


C Sources Equivalence Classfumaratemalatedeoxyuridine3-(3-hydroxyphenyl)propionateD-fructuronatesuccinatelactoseL-fucose2-oxoglutarate2-dehydro-3-deoxy-D-gluconateL-tartrateD-fructosetrehaloseD-mannoseD-galactitolarbutin3-phenylpropionateD-glucarateD-gluconateL-galactonateglyoxylatecitratemannosylglycerateL-idonateacetateL-ascorbate2,3-diketo-L-gulonate (C)

L-lyxose5-ketogluconateD-galactaratebeta-D-glucoseacetoacetatepsicoselysineglycerolbeta-D-ribopyranoseD-alloseD-sorbitolsalicinD-mannitoluridineD-galacturonatebeta-D-galactoseglycolateD-xyloseL-rhamnoseD-glucuronatethymidineD-galactonatemelibioseL-lysine


N Sources Equivalence Class

L-valinenitriteNH4+pyridoxamineL-phenylalanineL-tyrosineL-leucineL-isoleucinecytosine


CN Sources Equivalence ClasscytidinedeoxycytidineL-prolineputrescineL-serineglycine4-aminobutyratecyanatexanthosineN-acetylmuramateglucosamineL-argininephenylethylamineGlcNAc-1,6-anhMurNAc-L-Ala-gamma-D-Glu-DAP-D-AlaGlcNAc-1,6-anhMurNAcxanthineD-serine1,6-anhydro-N-acetylmuramate

L-ornithineL-glutamineN-acetyl-D-glucosaminechitobioseinosineD-alanineN-acetylneuraminateL-glutamateorotateL-asparagineL-threonineL-tryptophandeoxyinosinedeoxyadenosineadenosineL-aspartateL-alanine


Summary

Pathway/Genome DatabasesMetaCyc non-redundant DB of literature-derived pathways400 organism-specific PGDBs available through SRI at BioCyc.orgComputational theories of biochemical machinery

Pathway Tools softwareExtract pathways from genomesMorph annotated genome into structured ontologyDistributed curation tools for MODsQuery, visualization, WWW publishing


Acknowledgements

SRISuzanne Paley, Ron Caspi, Ingrid Keseler, Carol Fulcher, Markus Krummenacker, Alex Shearer, Tomer Altman, Joe Dale, Fred Gilham, Pallavi Kaipa

EcoCyc CollaboratorsJulio Collado-Vides, Robert Gunsalus, Ian Paulsen

MetaCyc CollaboratorsSue Rhee, Peifen Zhang, Kate DreherLukas Mueller, Anuradha Pujar

Funding sources:NIH National Institute of General Medical SciencesNIH National Center for Research Resources

BioCyc.org

Learn more from BioCyc webinars: biocyc.org/webinar.shtml

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Metabolic Network Analysis Algorithms in Pathway · PDF fileAlgorithms in Pathway Tools Peter D. Karp, Ph.D. Bioinformatics Research Group. ... Inference tools z Analyzing biological