Metabolic capacity of Bacillus subtilis for the production of purine nucleosides, riboflavin, and folic acid

We developed a stoichiometric model of Bacillus subtilis metabolism for quantitative analysis of theoretical growth and biochemicals production capacity. This work concentrated on biochemicals that are derived from the purine biosynthesis pathway; inosine, guanosine, riboflavin, and folic acid. These are examples of commercially relevant biochemicals for which Bacillus species are commonly used production hosts. Two previously unrecognized, but highly desirable properties of good producers of purine pathway-related biochemicals have been identified for optimally engineered product biosynthesis; high capacity for reoxidation of NADPH and high bioenergetic efficiency. Reoxidation of NADPH, through the transhydrogenase or otherwise, appears to be particularly important for growth on glucose, as deduced from the corresponding optimal carbon flux distribution. The importance of cellular energetics on optimal performance was quantitatively assessed by including a bioenergetic efficiency parameter as an unrestricted, ATP dissipating flux in the simulations. An estimate for the bioenergetic efficiency was generated by fitting the model to experimentally determined growth yields. The results show that the maximum theoretical yields of all products studied are limited by pathway stoichiometry at high bioenergetic efficiencies. Simulations with the estimated bioenergetic efficiency of B. subtilis, growing under glucose-limiting conditions, indicate that the yield of these biochemicals is primarily limited by energy and thus is very sensitive to the process conditions. The maximum yields that can reasonably be expected with B. subtilis on glucose were estimated to be 0.343, 0.160, and 0.161 (mol product/mol glucose) for purine nucleosides, riboflavin, and folic acid, respectively. Potential strategies for improving these maximum yields are discussed.