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Development of a Database Tool for Novel Biosynthetic Pathway Design

Kristala Jones Prather, Amanda Lanza, and Wei Chan. Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave, Room 66-458, Cambridge, MA 02139

Advances in the development of methods and methodologies for engineering biological systems have made it possible to envision the widespread use of living cells, especially microbes, as chemical factories. Methods such as directed evolution enable the production of a wide range of biological catalysts with varying substrate specificities while retaining defined functional group transformations. The tools of metabolic engineering allow one to functionally assemble multiple enzymes into a single host and to analyze and optimize the system for maximal production of the metabolite of interest. Combining the practices of biocatalysis and metabolic engineering should allow the design and construction of microbial cells capable of producing a variety of non-natural products. Indeed, we should imagine “retrobiosynthetic” design principles that are analogous to those of organic chemical synthesis. Design of these novel pathways requires the identification of enzymes capable of performing the desired functional group transformations. While several databases exist that provide access to detailed information about enzymes and known substrates, accessing this information based only on the functional group that has been transformed is not easily accomplished. We have begun the development of a new web-based database that defines the substrates and products only in terms of the functional groups that are transformed. This defines the base-level enzyme(s) that may be evolved to act upon a particular substrate, from which a specific enzyme-substrate-product group can be chosen. We are also incorporating estimated free energies associated with each transformation to enable rapid evaluation of the proposed pathway energetics. This database should facilitate the design of novel biosynthetic pathways, particularly through a retrobiosynthetic approach.