Tools for Modeling Metabolism in Mechanistic Enzymology

1. Tools for modeling metabolism Guest Lecture Graduate level course MCB221b - Mechanistic Enzymology Tobias Kind UC Davis Genome Center - MetabolomicsNovember 2007 • How far are we away (light years or light seconds?) • How we apply reductionist concepts for modeling metabolism • Computer tools and in-silico models This document is hyperlinked (pictures and green text). To use WWW links in this PPT switch to slide show mode.

2. Source: Nancy Moran In silico modeling of complex systems • What is a complex system? • Ant hill, a cell, humans,Candidatus Carsonella ruddii • (smallest genome n=182) • What means - to model? •  Select level of complexity: Philosophical description, molecular level, model dynamics,mathematical equations, implementation of algorithm • Problems? •  Non existing input variables; 2D metabolic pathways (reduced interactions); • lack of good software; model output can become extremely complex Source: WIKI Candidatus Carsonella ruddiisymbiont in plant lice (yellow)

3. Reductionism vs. Holism and Emergence Holism Reductionism Aristotle in the Metaphysics: "The whole is more than the sum of its parts." Emergence Descartes held that non-human animals could be reductively explained as automata — De homines 1622. Emergence refers to the way complex systems and patterns arise out of a multiplicity of relatively simple interactions. Source: ALL WIKIPEDIA

4. Metabolic engineering/molecular breeding • Alterations in metabolism by A) classical breeding B) genetic engineering • MIX : breeders use colchicine to double chromosomes and produce tetraploid plants (breeders become are genetic engineers) Source: UC Davis News Source: WIKI Breeder + German Giant (obtained by breeding) Ernesto and Titan Ted @ UC Davis Titan arum in Stuttgart (Amorphophallus titanum) click for more gardening  cultivating  breeding Polyploid gigantism, tetraploid gigantism

5. Modeling complex (bio)-chemical systems • Generic all purpose environments: • can model or simulate almost everything (like a space shuttle, traffic control) • Rational Rose Real Time (IBM), Mathematica, MatLab-Simulink and others • use markup languages (XML) like Unified Modeling Language (UML) • Specialized simulation environments: for biochemistry or systems biology • rely on biological data like pathway maps • use kinetic databases and enzyme model databases • use a specialized markup language like Systems Biology Markup Language (SBML) • A) Stoichiometry models – use reaction based system with stoichiometric matrix • B) Kinetic models – use kinetics data and stoichiometry data (in vivo/in vitro data differs) • C) Hybrid models use a combined subset of several (other) approaches

6. Metabolic engineering approaches • Metabolic control analysis (MCA) – mathematical framework how enzymes control or obtain an optimal flux • Metabolic flux analysis (MFA)- quantifies metabolic fluxes • Metabolic pathway analysis (MPA) – investigates entire flux distributions • Stochastic models – using random generators and Monte-Carlo techniques to cover all possible concentration ranges (high/low/mixed) for more realistic models • Deterministic models – using discrete concentrations and kinetic equations for being as accurate as possible FLUX - Rate of turnover of molecules through a reaction pathway. Elasticity coefficients – epsilon(i) = d ln(v(i)/d ln(p) ; v(i) is the rate of the enzyme in question and p is the parameter of the perturbation.

7. SBML (Systems Biology Markup Language) Source: Akira Funahashi – Cell Designer Tutorial • List of supported SBML programs (more than 200) from sbml.org • List of curated and published SBML models (around 200) from biomodels DB

8. Mathematical description representing a metabolic pathway • Aim: simulate the dynamic behavior of the intracellular compounds of a single cell • mass balances for each compound, i. e. reactions producing and consuming the compound; • enzyme kinetic equations that represent the rate of production and consumption of the compounds; • transport equations for uptake and excretion of substrate and products. Ċ - derivative of the compound concentrations with respect to time. N - stoichiometric matrix v - the vector of kinetic rate equations C -the vector of intracellular and extracellular compounds E - the vector of enzyme concentrations, P - the kinetic parameters of the rate equations. MMT - A pathway modeling tool for data from rapid sampling experiments Jochen Hurlebaus, Arne Buchholz, Wolfgang Alt, Wolfgang Wiechert and Ralf Takors,* In Silico Biology 2, 0042 (2002)

9. CellDesigner • Structured diagram editor for drawing gene-regulatory and biochemical networks • Uses Systems Biology Markup Language (SBML), a standard for representing models of biochemical and gene-regulatory networks. • Networks are able to link with simulation and other analysis packages through Systems Biology Workbench (SBW) • Runs under LinuX, WIN, MacOS • Connects to large repositories (BioModels, CellML and other) which providepre-defined models and reaction networks Download CellDesigner

10. CellDesigner - exchange of GTP for GDP

11. CellDesigner simulation result

12. Virtual Cell • Open source model (plug-in architecture) supported by NIH • Modeling and simulation of biochemical pathways coupled with electrophysiology, membrane transport, and diffusion/advection • Workflow for modeling and simulation which abstracts unnecessary complexity of the underlying math and physics • Around 200 models available (via server login) example: Kinetic analysis of receptor-activated phosphoinositide turnover RUN VCell with JAVA Webstart

13. JWS Online – kinetic models and MCA online • simulation of multiple kinetic models for several organisms • simulation of enzyme rates or metabolite concentrations • metabolic control analysis (MCA) • direct JAVA webstart out of web browser, client server model Start JWS Online

14. COPASI (GEPASI) • Stochastic and deterministic time course simulation • Steady state analysis (including stability) • Metabolic control analysis / sensitivity analysis • Elementary mode analysis; Mass conservation analysis • Import/export of SMBL, can create C source code Download Copasi

15. General use of in-silico models for metabolic engineering • Visualization and modeling of complex interactions can lead to better understanding • Idea generation + exploration of combinatorial space + design of experiment • Hypothesis testing and experimental validation of predictions Example (A): Increasing Bio-Ethanol productionAim: decrease glycerol content and increase ethanol yield by 10% Result: 40% decrease in glycerol and 3% increase in ethanol Approach: genome-scale metabolic model + gene insertion, gene deletion Example (B): Increase fermentation production of succinic acid Aim: increase succinic acid production Result: nine-fold increase of succinic acid Approach: genome and pathway analysis & flux analysis + combinatorial gene knockout A) In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production; Metabolic Engineering 8 (2006) 102–111 B) Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation; APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2005, p. 7880–7887

16. Flammarion (WIKI) Tools for modeling metabolism outside theSystems Biology world • Well established tools based on Qualitative Structure-Activity Relationships (QSAR) and model based algorithmsor rule base expert systems • Used for prediction of organic reactions, drug metabolism(cytochromes P450), environmental degradation and toxicity estimations, important for OECD large volume chemicals Source: TIMES and CATABOL program: OASIS BourgasSabcho Dimitrov, Ovanes Mekenyan in METABOLISM in-silico simulation

17. Colossus computer. Source (WIKI) Outlook • Standardized and open XML languages (SBML) most likely successful • Community driven tools most likely successful in the future becausenumber of people involved is high; data sharing of models important • Lots of room for commercial exploitation (better support) • Do not expect Artificial Intelligence aka Star Trek “Computer tell me…”expect to do your homework first, then use software models to model it. • More than 120 modeling programs exist, expect more to come. • The lack of super-integration of experimental datafrom transcriptomics, proteomics and metabolomicsexperiments currently hinders in-silico modeling and limits predictive space of developed in-silico models - hence the ultimate systems biology dream: complete modeling of metabolism, cells and whole organisms

18. Homework for homework discussion III (1h) • Play the game of life in JAVA (webstart) explain why it was a milestone development. • Name three problems during model implementation (for example: no stoechiometry data existing, only in vitro kinetics available) • Tell three reasons why we should be able to model simple a bug like Candidatus Carsonella ruddii(182 genes only!) • Tell three reasons why we are not able to model simple a bug like CC ruddii. • Download one of the tools discussed here or one from SBML.ORG and report on the usefulness. • Tell three reasons why metabolic engineering will solve the world hunger problem. • Tell three reasons why metabolic engineering will not solve the world hunger problem. • Name three vitamins and three drugs which can be obtained by metabolic engineering • Explain why successful metabolic engineering requires a systems biology approach. • +++ Additional question (after solving all others) +++You are the head of the newly founded Metab. Eng. Dept. in XYZ. You need to perform and present a strategic decision in front of the board of directors which three of the 120 tools from SBML.ORGwill be used during the next five years in your department (total fund of 300K is provided for that).

19. Reading List (all DOI linked) (25 min) Experimental and mathematical approaches to modeling plant metabolic networks Predictive Metabolic Engineering: A Goal for Systems Biology MMT - A pathway modeling tool for data from rapid sampling experiments Mathematical modelling of metabolism The production of fine chemicals by biotransformations Additional reading just for interest (prediction power of in-silico models): In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production Probing the performance limits of the Escherichia coli metabolic network subject to gene additions or deletions Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation Here is the evidence, now what is the hypothesis? The complementary roles of inductive and hypothesis-driven science in the post-genomic era

20. Link List 1 Substrate, competitive, uncompetitive, noncompetitive inhibition http://en.wikipedia.org/wiki/Enzyme_inhibitor Reductionism http://en.wikipedia.org/wiki/Reductionism Emergenz http://de.wikipedia.org/wiki/Emergenz Complex Systems and origin of life http://books.google.com/books?ct=title&psp=1&q=modeling+complex+systems+emergence Tools http://www.google.com/search?q=Tools+for+modeling+metabolism In vivo modeling http://www.google.com/search?hl=en&q=in+vivo+metabolic+modelling&btnG=Search MMT – tool for metabolic modeling http://www.bioinfo.de/isb/2002/02/0042/ MMT http://www.bioinfo.de/isb/gcb01/poster/hurlebaus.html ALSYSTA http://stephanopoulos.openwetware.org/ Tools search http://www.google.com/search?hl=en&q=ERATO%2C+BioSpice%2C+DBSolve%2C+E-Cell%2C+Gepasi%2C+Jarnac%2C+StochSim%2C+and+Virtual+Cell&btnG=Search Modeling cells and metabolism http://www.mas.ncl.ac.uk/~ncsg3/hackathon07/participants/ Colchicine and algae http://www.google.com/search?q=Susceptibility+of+Colchicum+and+Chlamydomonas+to+Colchicine

21. Link List 2 SBML model repository (for CellDesigner and others) http://www.systems-biology.org/001/ Systems Biology Markup Language (org) has hundreds of software tools listed http://sbml.org/index.psp US GOV network for metabolic engineering http://www.metabolicengineering.gov The ultimate goal of Systems Biology http://www.google.com/search?hl=en&q=%22ultimate+goal+of+systems+biology Stoechiometry models and power law models http://www.google.com/search?q=e-cell++gepasi+Stoichiometric+models Catabol, Meteor, Lhasa http://www.google.com/search?hl=en&q=catabol+metabolism+lhasa+meteor&btnG=Search Thank you!Thanks to Wikimedia!Thanks to the FiehnLab!