Regulating expression of pyruvate kinase in Bacillus subtilis for control of growth rate and formation of acidic byproducts.

Our prior work has shown that a pyk mutant of Bacillus subtilis exhibited diminished acidic byproduct accumulation, dramatically elevated phosphoenolpyruvate (PEP) pool, and reduced growth rate. To determine if a low acetate-producing but fast-growing strain of B. subtilis could be developed, we placed the expression of the pyk gene under the control of an inducible promoter. Enzyme measurements proved that PYK activity of the inducible PYK mutant (iPYK) increases with the isopropyl-beta-d-thiogalactopyranoside concentration. Batch growth experiments showed that growth rate and acid formation are closely related to the induction level of pyk. Measurements of cell growth rate and acetate formation of the iPYK mutant at different induction levels revealed that a PYK activity of about 12% of wild-type allows for good growth rate (0.4 h(-)(1) versus 0.63 h(-)(1) of wild-type) and low acetate production (0.26 g/L versus 1.05 g/L of wild-type). This is the first report to our knowledge of a metabolically engineered B. subtilis strain that allows good growth rate and low acid production in batch cultures. Finally, it was found that, by varying the pyk induction level, intracellular PEP concentration can be controlled over a wide range. The intracellular PEP concentration is intimately connected to the regulation of the transport of phosphotransferase system (PTS) sugars in the presence of glucose. Because there is no other method for modulating intracellular PEP levels, this finding represents a major advance in one's ability to dissect the function of the PTS and sugar metabolism in bacteria.