Fermentation patterns of Escherichia coli with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the Rhizobium etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99A-pyc than by cells which overproduced PPC (JCL1242/pPC201, ppc ؉ ), even though PPC activity in cell extracts of JCL1242/pPC201 (ppc ؉ ) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism the pyc ؉ strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the ppc ؉ strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation.In mixed-acid fermentation of glucose, succinate is formed via the reductive arm of the tricarboxylic acid cycle, a pathway which includes the fixation of 1 mol of carbon dioxide per mol of succinate generated. The key reaction in this sequence is the carboxylation of three-carbon intermediates such as phosphoenolpyruvate (PEP) to four-carbon oxaloacetate. The principal PEP-carboxylating enzyme found in Escherichia coli is PEP carboxylase (PPC). In E. coli PEP may also be converted to pyruvate, which during anaerobic growth leads to the formation of lactate, formate, acetate, and ethanol. In other prokaryotes and many eukaryotes during glucose metabolism, oxaloacetate is synthesized by carboxylation of pyruvate by pyruvate carboxylase (PYC) (3,24,25), an enzyme that is absent in E. coli. PEP is also required for glucose consumption via the PEP-phosphotransferase system (PEP-PTS) and for the synthesis of aromatic amino acids (7,14). Because of its central position in glucose metabolism, PEP partitioning is highly regulated by cellular mechanisms.In order to affect the metabolic rigidity of the biochemical network at the PEP branch point, several metabolic engineering approaches have been proposed. As one would expect, increased succinate production has been shown to result from overexpression of PPC from E. coli (20) or overexpression of PYC via the Rhizobium etli pyc gene (13). Similarly, the expression of malic enzyme in E. coli strains lacking the enzymes pyruvate formate lyase (PFL) and lactate dehydrogenase (LDH) yielded succinate as the major fermentation product (28). Each of these genet...

Oxaloacetate (OAA) plays an important role in the tricarboxylic acid cycle and for the biosynthesis of a variety of cellular compounds. Some microorganisms, such as Rhizobium etli and Corynebacterium glutamicum, are able to synthesize OAA during growth on glucose via either of the enzymes pyruvate carboxylase (PYC) or phosphoenolpyruvate carboxylase (PPC). Other microorganisms, including Escherichia coli, synthesize OAA during growth on glucose only via PPC because they lack PYC. In this study we have examined the effect that the R. etli PYC has on the physiology of E. coli. The expressed R. etli 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 an E. coli ppc null mutant in minimal glucose medium, and PYC expression caused increased carbon flow towards OAA in wild-type E. coli cells without affecting the glucose uptake rate or the growth rate. During aerobic glucose metabolism, expression of PYC resulted in a 56% increase in biomass yield and a 43% decrease in acetate yield. During anaerobic glucose metabolism, expression of PYC caused a 2.7-fold increase in succinate concentration, making it the major product by mass. The increase in succinate came mainly at the expense of lactate formation. However, in a mutant lacking lactate dehydrogenase activity, expression of PYC resulted in only a 1.7-fold increase in succinate concentration. The decreased enhancement of succinate formation in the /dh mutant was hypothesized to be due to accumulation of pyruvate and NADH, metabolites that affect the interconversion of the active and inactive form of the enzyme pyruvate formate-lyase.

No abstract

The production of organic acids by two anaerobic ruminal bacteria Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD-1, was compared with glucose, cellobiose, microcrystalline cellulose, Walseth cellulose (acid swollen cellulose), pulped paper, and steam-exploded yellow poplar as substrates. The major end product produced by F. succinogenes from each of these substrates was succinate (69.5-83%), the principal secondary product was acetate (16-30.5%). Maximum succinate productivity ranged from 14.1 mg/L.h for steam-exploded yellow Poplar to 59.7 mg/L.h for pulped paper. For R. flavefaciens, the major end product from cellobiose, microcrystalline cellulose, and acid-swollen Walseth cellulose was acetate (39-46%), pulped paper and steam-exploded yellow poplar yielded succinate (42-54%) as the major product. Maximum succinate productivity by R. flavefaciens ranged from 9.21 mg/L.h for cellobiose to 43.1 mg/L.h for pulped paper. In general, much less succinate was produced at a lower maximum productivity by R. flavefaciens than by F. succinogenes under similar fermentation conditions. The maximum succinate productivities by these two organisms are comparable to the previously reported value of 59 mg/L.h for Anderobiospirillum succiniciproducens grown on glucose and corn steep liquor.

No abstract