1. SRI International Bioinformatics 1 A BRG Biofuels Metabolic Engineering Project Bioinformatics Research Group SRI International Caspi@ai.sri.com Latendre@ai.sri.com Pkarp@ai.sri.com Ron Caspi Mario Latendresse Peter Karp October 2010 Pathway Tools Workshop

2. SRI International Bioinformatics 2 Two Main Parts This project will be conducted in the BRG group. Funded by the Department of Energy (DOE) under the direction of Peter Karp, PI Soon to be started The project has two main parts: 1. Curating MetaCyc for biofuel pathways under the supervision of Ron Caspi, co-PI; 2. A computational tool to infer new pathways, co-PI, Mario Latendresse. (Not biofuels specific)

3. SRI International Bioinformatics 3 Computational Tool Basic statement of the problem: Given a source and target metabolites, what are the possible metabolic pathways from the source to the target in a given organism? And rank these pathways based on their quality And constrained these pathways: number of carbon atoms from the source to the target, length of pathway, number of genes to delete or add, and so on

4. SRI International Bioinformatics 4 Computational Tool Approach Use a searching algorithm (A*) that can use expert knowledge as well as precise evaluation algorithms of the quality of pathways found Based on 1. Tracing of carbon atoms in pathways. From the source to target, the number of carbon atoms can be evaluated. 2. Gibbs free energy of reactions will help the evaluation of the feasibility of existing and new pathways. A prototype A*, based on compound similarity, has been written to find the shortest pathway between two compounds

5. SRI International Bioinformatics 5 The A* algorithm A general searching algorithm in a tree-like search space Uses two evaluating functions g(path) and h(rest- path) l Function g evaluates the path (length, Gibbs energy, cost of enzymes, and so on) seen so far l Function h evaluates the potential of current path (based on heuristics) to the target metabolite l Next node to search is based on the value of h: go in the best direction Function h can use expert knowledge as well as precise calculations

6. SRI International Bioinformatics 6 A* Searching

7. SRI International Bioinformatics 7 A* Prototype Results Found all the pathways described in the paper by McShan, Rao, Shah on Pathminer, Bioinformatics, Vol. 19, no 13, 2003. For example, from citrate to L-histidine (E. coli) In less than a second or a few seconds for a seven step pathways Proof of concept since the feasibility of such pathways is not proven: not enough constrained applied so far

8. SRI International Bioinformatics 8 Gibbs Free Energy Computation by the Group Contribution Method Prototype implementation of Jankowskis method Still needs a full evaluation

9. SRI International Bioinformatics 9 Curation of Biofuel Pathways in MetaCyc 1. Identify lignocellulose degrading enzymes and curate them into MetaCyc 2. Expand the coverage of hydrogenases in MetaCyc to include representatives of all known classes and subtypes 3. Expand MetaCyc coverage of fatty acids and lipids biosynthetic enzymes to reflect currently known metabolic and genetic information about oil production in algae and cyanobacteria 4. We will identify biochemically verified pathways involved in processes relevant to biofuel production

10. SRI International Bioinformatics 10 Graphical Extension of Pathway Tools Show complex polymers degradation processes that involve multiple enzymes attacking simultaneously a complex molecule at different sites

11. SRI International Bioinformatics 11 Validating the Computational Tool The new biofuel pathways will be used to evaluate the capability of the computational tool.

12. SRI International Bioinformatics 12 A BRG Biofuels Metabolic Engineering Project Questions?