Because the complexity of metabolism cannot be intuitively understood or analyzed, computational methods are indispensable for studying biochemistry and deepening our understanding of cellular metabolism to promote new discoveries. We used the computational framework BNICE.ch along with cheminformatic tools to assemble the whole theoretical reactome from the known metabolome through expansion of the known biochemistry presented in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We constructed the ATLAS of Biochemistry, a database of all theoretical biochemical reactions based on known biochemical principles and compounds. ATLAS includes more than 130 000 hypothetical enzymatic reactions that KEGG connect two or more KEGG metabolites through novel enzymatic reactions that have never been reported to occur in living organisms. Moreover, ATLAS reactions integrate 42% of KEGG metabolites that are not currently present in any KEGG reaction into one or more novel enzymatic reactions. The generated repository of information is organized in a Web-based database (http://lcsb-databases.epfl.ch/atlas/) that allows the user to search for all possible routes from any substrate compound to any product. The resulting pathways involve known and novel enzymatic steps that may indicate unidentified enzymatic activities and provide potential targets for protein engineering. Our approach of introducing novel biochemistry into pathway design and associated databases will be important for synthetic biology and metabolic engineering.