Bacillus subtilis is a gram positive bacteria often utilized in industry as a producer of high quality enzymes and proteins [1]. One of the primary challenges involved in the use of B. subtilis in industry is the extensive regulatory pathways in the cell, making the flux through the metabolic reactions of the cell extremely resistant to alteration by genetic manipulation [2]. It has already been demonstrated that removing a portion of the regulatory genes in B. subtilis results in significantly enhanced protein production by the cell [3]. Now, we are endeavoring to produce a minimal strain of the B. subtilis genome. This minimal strain will lack every dispensable alternative metabolic pathway and every dispensable regulatory gene, making the strain much more amenable to alteration for industrial use. B. subtilis was selected as the platform organism for the construction of this minimal strain because the natural competence of B. subtilis allows for rapid knockouts of single genes and intervals of genes [4]. Additionally, the extensive information available about B. subtilis will allow for a systematic and planned approach to be used during the construction of the minimal strain. To facilitate the development of our minimal organism, we have constructed a new genome-scale metabolic model of B. subtilis based on the annotations available in the SEED subsystems-based annotation environment [5] and supplemented by data included in two previously developed B. subtilis models [6, 7]. The new model includes elements of the B. subtilis regulation when necessary to properly predict the effect of gene knockouts. The thermodynamic properties of the model reactions were estimated to predict the reversibility of model reactions [8]. The new model also includes numerous genes and pathways not found in any previous models. The new model was validated against a variety of experimental observations including biolog phenotyping array results [7], gene essentiality data [9], 60 published gene interval knockout experiments [3], and over 300 new gene-interval knockout experiments. When model predictions did not match experimental observations, a variety of methods were developed and applied to improve the model accuracy. As a result of these efforts and the inclusion of features from currently published models, this is the most complete and accurate metabolic model of B. subtilis constructed to date. We will discuss the methods used to assemble and correct this new genome-scale model; we will compare the accuracy and content of the new model with previously published models; and we will discuss the application of the model to the design of a systematic and optimized strategy for combining gene interval knockouts to produce a minimal strain of B. subtilis.

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