The physiological effects and metabolic alterations caused by the expression of Rhizobium etli pyruvate carboxylase in Escherichia coli

Oxaloacetate (OAA) plays an important role in the tricarboxylic acid cycle and for the biosynthesis of a variety of cellular compounds. Some microorga nisms, such as Rhizobium etli and Corynebacterium glutamicum, are able to s ynthesize OAA during growth on glucose via either of the enzymes pyruvate c arboxylase (PYC) or phosphoenolpyruvate carboxylase (PPC). Other microorgan isms, including Escherichia coli, synthesize OAA during growth on glucose o nly via PPC because they lack PYC. In this study we have examined the effec t that the R. etli PYC has on the physiology of E. coli. The expressed R. e di PYC was biotinylated by the native biotin holoenzyme synthase of E. coli and displayed kinetic properties similar to those reported for alpha4 PYC enzymes from other sources. R. etli PYC was able to restore the growth of a n E. coli ppc null mutant in minimal glucose medium, and PYC expression cau sed increased carbon flow towards OAA in wild-type E. coli cells without af fecting the glucose uptake rate or the growth rate. During aerobic glucose metabolism, expression of PYC resulted in a 56% increase in biomass yield a nd a 43% decrease in acetate yield. During anaerobic glucose metabolism, ex pression of PYC caused a 2.7-fold increase in succinate concentration, maki ng it the major product by mass. The increase in succinate came mainly at t he expense of lactate formation. However, in a mutant lacking lactate dehyd rogenase activity, expression of PYC resulted in only a 1.7-fold increase i n succinate concentration. The decreased enhancement of succinate formation in the ldh mutant was hypothesized to be due to accumulation of pyruvate a nd NADH, metabolites that affect the interconversion of the active and inac tive form of the enzyme pyruvate formate-lyase.