METABOLIC CAPACITY OF BACILLUS-SUBTILIS FOR THE PRODUCTION OF PURINE NUCLEOSIDES, RIBOFLAVIN, AND FOLIC-ACID

We developed a stoichiometric model of Bacillus subtilis metabolism fo r quantitative analysis of theoretical growth and biochemicals product ion 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 biochemica ls for which Bacillus species are commonly used production hosts. Two previously unrecognized, but highly desirable properties of good produ cers of purine pathway-related biochemicals have been identified for o ptimally engineered product biosynthesis; high capacity for reoxidatio n of NADPH and high bioenergetic efficiency. Reoxidation of NADPH, thr ough the transhydrogenase or otherwise, appears to be particularly imp ortant for growth on glucose, as deduced from the corresponding optima l carbon flux distribution. The importance of cellular energetics on o ptimal performance was quantitatively assessed by including a bioenerg etic efficiency parameter as an unrestricted, ATP dissipating flux in the simulations. An estimate for the bioenergetic efficiency was gener ated by fitting the model to experimentally determined growth yields. The results show that the maximum theoretical yields of all products s tudied are limited by pathway stoichiometry at high bioenergetic effic iencies. 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 i s very sensitive to the process conditions. The maximum yields that ca n 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 nucle osides, riboflavin, and folic acid, respectively. Potential strategies for improving these maximum yields are discussed. (C) 1998 John Wiley & Sons, Inc.