SUPPRESSED ACID FORMATION BY COFEEDING OF GLUCOSE AND CITRATE IN BACILLUS CULTURES - EMERGENCE OF PYRUVATE-KINASE AS A POTENTIAL METABOLIC ENGINEERING SITE

Microbial cultures typically produce acids when metabolizing the commo n carbon source, glucose. Acid production not only represents a waste of carbon, but its accumulation can limit cell concentration and cultu re stability, thereby reducing productivity. On the basis of prior wor k, acid production was attributed to be due to a mismatch between glyc olytic and tricarboxylic acid (TCA) cycle capacities. To suppress acid production, a strategy entailing adding citrate to glucose minimal me dium proved extremely effective. The effect of citrate on in-vivo flux distribution was quantified using a detailed flux-model. When the mol ar glucose-citrate ratio was varied between 3 and 6, a significant red uction in glycolytic flux and essentially complete suppression of acid formation was found as compared to chemostat cultures grown solely on glucose. Adding other biosynthetic precursors such as glutamine did n ot invoke the same suppression, thus indicating that citrate's effect is at the regulatory level. We hypothesized that the reduction of glyc olytic flux in the presence of citrate results from its transport bein g coupled with the uptake of divalent metal ions. Citrate transport al ters the intracellular balance of metal ions which in turn could trigg er a sophisticated series of metabolic events leading to reduction of the activities of the pyruvate kinase and phosphofructokinase (PFK), t he regulatory enzymes of glycolysis. On the basis of this scenario and other regulatory information, pyruvate kinase has emerged as a potent ial metabolic engineering site. It's deactivation in Bacillus subtilis or Escherichia coli strains is expected to yield constructs with a mu ch lower tendency for making acid byproducts.