Microbial cultures usually produce a significant amount of acidic byproducts which can repress cell growth and product synthesis. Metabolic engineering provides a powerful approach to optimize the cellular activities and improve product yields by genetically manipulating specific metabolic pathways. In this study, an inducible PYK (iPYK) mutant of B. subtilis was constructed based on the outcome of metabolic modeling. The results demonstrate that good cell growth rate and low acetate formation can be attained at an appropriate PYK expression level. In Addition, the PYK mutation was explored as an efficient way to improve the production of recombinant protein and valuable metabolites such as folic acid. A combination of PYK mutation and additional mutations in the folic acid synthesis pathway resulted in an 8-fold increase in folic acid production. Recombinant protein was improved two-fold by the PYK mutation due to low acetate formation and longer production time in the pyk mutant.