W132 Nucleic Acids Research, 2009, Vol. 37, Web Server issue Construction of reaction matrix Information on reactions and enzymes was obtained from KEGG pathway maps and the equations of each reaction were determined. Therefore, a reaction matrix was con- structed based on maps, reactions and enzymes data. For instance, ‘R01731: Oxaloacetate + L-Arogenate <=> L-Aspartate + Prephenate’ is converted into ‘R01731: C00036 + C00826 <=> C00049 + C00254’ (where R01731 is KEGG reaction ID; C00036, C00826, C00049 and C00254 are KEGG compound ID). The reac- tion R01731 is a reversible reaction which is regarded as two forward reactions in FMM. The direction of one for- ward reaction is from C00254 to C00036 in the pathway map, the other is from C00036 to C00254. The example illustrate herein is only one forward reaction from C00254 to C00036. Therefore, four reactions are described below: C00254 => C00826 C00254 => C00036 C00049 => C00826 C00049 => C00036 1 2 3 4 Each equation corresponds to an edge in a graph; Figure S1 (See Supplementary Data) depicts an example reaction matrix. FMM contains 16884 compounds; and so the size of the reaction matrix is 16884 16884. A direct path graph of the reactions depends on the reaction matrix using the breadth ﬁrst search (BFS) approach. As shown in Figure S2 (See Supplementary Data), when the direct path graph from compound C00254 to compound C00079 is iden- tiﬁed by BFS, the search terminates when the C00079 node is found. Additionally, KEGG MODULE is a new collection of pathway modules, molecular complexes, and other func- tional units, each represented as a list of KEGG Orthology (KO) identiﬁers, which were used in pathway reconstruction when the node was a KEGG MODULE. Reconstruction of metabolic pathway from various KEGG pathway maps After all possible reaction paths were identiﬁed, the number of pathway maps was calculated. Usually, found paths occurred not in only a single pathway map, but in a complicated fashion in several maps. Pathway maps that contain the most paths are selected and the one pathway map that has only one reaction is avoided. A matrix of maps versus reactions was employed to reconstruct meta- bolic pathway from diﬀerent KEGG maps. For example, the pathway from C00254 to C00811 passes through sev- eral reactions (paths) and maps, as described in Figure S3 (See Supplementary Data). It is better to select fewer maps and fewer map exchanges to reconstruct metabolic pathway. Therefore, based on the above example, ‘map00400 ! map00400 ! map00360 ! map00360’ is much better than ‘map00400 ! map00400 ! map00960 ! map00360’. COMPARATIVE ANALYSIS Comparative analysis provided in FMM is useful in synthetic biology. For example, the reconstruction of metabolic pathways to produce valuable metabolites or secondary metabolites in bacteria or yeast is a promising biotechnological strategy. Comparative analysis provides an easy way to elucidate which genes from which species should be cloned into those microorganisms. First, the enzymes identiﬁed in the reconstructed pathway were processed to search for orthologous encoding genes from various species. Then, the presence or absence of the path- way in a particular species can be known. Furthermore, comparative tables of several organisms are provided. The species are ranked according to the numbers of enzymes involved in the reconstructed pathway. WEBSITE USAGE Web Interface Figure S4 (See Supplementary Data) presents the user- friendly web interface of the FMM. In ‘Start FMM’, two metabolites of interest (keyword or KEGG com- pound ID) can be input and several species can be selected from four categories (animals, plants, fungi and prokar- yotic). The metabolic pathways corresponding to the two input metabolites can be reconstructed, as given in Figure S4A (See Supplementary Data). As to the compar- ative analysis of metabolic pathways, Figure S4B (See Supplementary Data) includes one species in the ‘major species’ column (of microorganisms commonly used in synthetic biology) and several in the ‘comparative species’ column (of several organisms in common use in the laboratory). The comparative metabolic pathways between various organisms are then generated. The out- puts include two views of the metabolic pathway; one is a vertical view (local view); the other highlights the meta- bolic pathway in the KEGG map (global view), as shown in Figure 3A and B, respectively. The EC number and hyperlinks to detailed information (such as orthologous enzymes and PTM) of the enzymes that are involved in the pathway are supplied when the names of the enzymes and star symbols in the output table and ﬁgure are clicked. When the metabolic pathway traverses more than one KEGG map, it can be connected from several KEGG maps using our system, as presented in Figure S5 (See Supplementary Data). Case studies Case study I—application in plant secondary metabolites production. Plant-speciﬁc ﬂavonoids are important sec- ondary metabolites which are synthesized from 4-coumaroyl coenzyme A (CoA) and derived from the general phenylpropanoid pathway, and three malonyl- CoAs. Numerous plant ﬂavonoid biosynthetic enzymes, which are individually expressed in E. coli as functional enzymes, have been identiﬁed (26,27). Furthermore, Watts et al. (28) successfully cloned several critical genes from plants into E. coli and observed the high-level production of ﬂavanone naringenin by feeding exogenous 4-coumarate. In this work, the system of Watts et al.’s was adopted as an example to demonstrate the eﬀective- ness of FMM. When either the genes that can be cloned into E. coli to produce naringenin from 4-coumarate,