| Microbial cultures usually produce a significant amount of acidic byproducts which can repress cell growth and product synthesis. In addition, the production of acids is a waste of carbon source thereby reducing the product yield and productivity.; Metabolic engineering provides a powerful approach to optimize the cellular activities and improve product yields by genetically manipulating specific metabolic pathways. Previous work has identified mutation of pyruvate kinase (PYK) as an efficient way to reduce acid production; however, complete abolishment of PYK in Bacillus subtilis resulted in dramatically reduced cell growth rate. In this study, an inducible PYK (iPYK) mutant of B. subtilis was constructed and extensively characterized. The results demonstrate that good cell growth rate and low acetate formation can be attained at an appropriate PYK expression level. In addition, mutation of phosphofructokinase (PFK) in the glycolysis pathway also provides an alternative approach to reduce acetate formation.; Two outcomes of the pyk mutant of B. subtilis , high phosphoenolpyruvate (PEP) pool and low acetate concentration, prompted us to investigate the employment of the pyk mutation as an efficient way to improve folic acid and recombinant protein production. The high intracellular PEP and glucose-6-phosphate (G6P) concentrations in the pyk mutant led to higher folic acid production by providing abundant synthetic precursors. Additional mutations in the folic acid synthesis pathway, along with the pyk mutation, 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. In addition, using glycerol instead of glucose as the carbon source reduced acetate production and improved protein production by 60%.; The effect of citrate on acetate production in Bacillus thuringiensis (Bt) cultures was investigated and the continuous culture results show that citrate is very effective at reducing acetate formation. These results indicate that pyk may be a potential mutation target for reducing acetate formation in Bt.; Descriptors. Acetate, Bacillus subtilis, Bacillus thuringiensis, Byproduct, Folic Acid, Glucose-6-phosphate, Glycolysis, Metabolic Engineering, Phosphoenolpyruvate, Phosphofructokinase, Pyruvate Kinase, Recombinant Protein... |
