Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvatekinase flux

In this study, it is found that, for Bacillus subtilis, citrate-glucose com etabolism leads to zero acid production over a wide range of growth rates a nd nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic fl ux attenuation. First, the measured fluxes show that, compared with culture s grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolp yruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maxim izing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review sugges ts some mechanisms for how PYK attenuation by citrate addition can occur. A t this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP po ol. These two inhibition events combine to throttle glycolytic flux; minimi ze acid formation; and substantially increase cellular, product, and energe tic yields. (C) 1999 John Wiley & Sons, Inc.