Physiologic consequences of glucose transport and phosphoenolpyruvate node modifications in Bacillus subtilis 168

The phosphoenolpyruvate (PEP) node is an important carbon distribution point in the central metabolic networks; therefore, its modification is a common strategy employed for developing microbial production strains. In this study, mutants of Bacillus subtilis 168 were generated with deletions of pykA (which encodes pyruvate kinase), ptsG (which encodes the glucose-specific IICBA(Glc) component) or the ptsGHI operon [which encodes IICBA(Glc), HPr protein and enzyme I from the PEP:sugar phosphotransferase system (PTS)]. These modifications caused a reduction in the initial rate of [(14)C]-glucose import, corresponding to 10.99, 2.83 and 0.50% of that found in B. subtilis 168 for strains with inactive pykA, ptsG or ptsGHI genes, respectively. Characterization of derivative strains lacking 3-dehydroquinate synthase activity showed that inactivation of pykA leads to an 8-fold increase in carbon flow to the common aromatic pathway. Quantitative real-time PCR analyses of 76 genes from several functional classes revealed a carbon starvation transcriptional pattern that includes a partial gluconeogenic response and overexpression of genes encoding non-PTS glucose importers in the strains lacking functional pykA, ptsG or ptsGHI genes. A transcriptional response consistent with pyruvate limitation was also detected, which includes upregulation of genes encoding malic enzymes that generate pyruvate from malate.