Metabolic Analysis of Escherichia coli in the Presence and Absence of the Carboxylating Enzymes Phosphoenolpyruvate Carboxylase and Pyruvate Carboxylase

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.     

Effects of phosphoenol pyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum

The phosphoenol pyruvate carboxylase gene (ppc) of lysine-producing Corynebacterium glutamicum and C. lactofermentum strains was inactivated by marker exchange mutagenesis. The mutants lacked completely phosphoenol pyruvate carboxylase (PEP carboxylase) activity, but grew in minimal medium containing glucose as the sole carbon source. In addition, the ppc ^– strains produced equivalent titers of lysine in shake flasks and in 10-l fermentation experiments as their parent strains. To address the question of how ppc ^– Corynebacterium strains generate oxaloacetate (OAA) for their own metabolism as well as for high-level lysine production, we measured the activities of enzymes leading to OAA synthesis. Whereas pyruvate carboxylase activity was not detected in any of the strains, phosphoenol pyruvate carboxykinase (PEP carboxykinase) activity was found to be significantly higher in C. glutamicum ppc mutants compared to the parent strains. On the other hand, PEP carboxykinase activity in C. lactofermentum was essentially absent. As glyxylate cycle enzymes are strongly repressed by glucose, they are not likely to compensate for the lack of PEP carboxylase activity. PEP carboxykinase, among several candidates, could play this role. Correspondence to: M. Gubler     

Metabolic analysis of Escherichia coli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase

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.     

Detection of genetic variation in Bradyrhizobium japonicum USDA 110 variants using DNA fingerprints generated with GC rich arbitrary PCR primers

Bradyrhizobium japonicum USDA 110 has been shown to contain several genetically similar naturally occurring colony morphology variants. These variants differ in symbiotic nitrogen fixation ability and in the utilization of various carbon substrates. They have been shown to share extensive DNA homology and appear to be derived from a common ancestor. Despite these similarities certain B. japonicum USDA 110 variants have been shown to be devoid of symbiotic nitrogen fixation. One of these variants (L2-110), however, was recently shown to possess significant levels of explanta nitrogen fixation and to synthesize functional dinitrogenase enzyme within bacteroids. In an effort to identify genetic markers which could explain differences in symbiotic nitrogen fixation between B. japonicum variants, DNA fingerprints were generated by PCR using arbitrary primers. Two of these primers with GC rich sequences were able to differentiate between B. japonicum USDA 110 variants I-110, L2-110, and MN-110. Unique markers have now been identified which could be examined further to determine if they explain the differences in symbiotic nitrogen fixation between USDA 110 variants.     

Yeast pyruvate carboxylase: identification of two genes encoding isoenzymes

In Saccharomyces cerevisiae, pyruvate carboxylase [EC 6.4.1.1] has an important anaplerotic role in the production of oxaloacetate from pyruvate. We report here the existence of two pyruvate carboxylase isozymes, which are encoded by separate genes within the yeast genome. Null mutants were constructed by one step gene disruption of the characterised PYC gene in the yeast genome. The mutants were found to have 10-20% residual pyruvate carboxylase activity, which was attributable to a protein of identical size and immunogenically related to pyruvate carboxylase. Immunocytochemical labelling studies on ultrathin sections of embedded whole cells from the null mutants showed the isozyme to be located exclusively in the cytoplasm. We have mapped the genes encoding both enzymes and shown the previously characterised gene, designated PYC1, to be on chromosome VII whilst PYC2 is on chromosome II.     

Significance of Pyruvate Carboxylase in Sugar Metabolism of Agrobacterium tumefaciens

Mutants, which fail to grow on glucose medium but can grow on succinate medium, were isolated by treatment with N-methyl-N′-nitro-N-nitrosoganidine from the wild-type strain of Agrobacterium tumefaciens, and were found to lose growth on several hexoses and three-carbon intermediates. The revertant mutants, which recovered the ability to grow on glucose medium, simultaneously regained the ability to grow on hexoses and three-carbon intermediates. By comparison of biochemical properties of the wild-type, the mutants and the revertant mutants, two mutant strains were characterized to be pyruvate carboxylase-deficient. Then, we concluded that these mutants might be induced by a single mutation at a genetic locus of pyruvate carboxylase and that the deficiency in the enzyme gave a pleiotropic effect on the ability to grow on hexoses and three-carbon intermediates. Some properties of pyruvate carboxylase of this bacterium were also presented.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Cloning of the gene for phosphoenolpyruvate carboxylase from Azotobacter chroococcum, an enzyme important in aerobic nitrogen fixation

Summary Azotobacter chroococcum Fos 189 is a Tn1-induced mutant which, unlike the parent strain MCD1, does not fix nitrogen in air when provided with glucose or pyruvate as sole carbon sources. Fos 189 showed 5% of parental activity for phosphoenolpyruvate carboxylase though PEP synthetase activity was normal. The A. chroococcum phosphoenolpyruvate carboxylase (ppc) gene was isolated after complementation of an appropriate Escherichia coli mutant using a broad host range gene bank prepared from A. chroococcum genomic DNA. The gene was localised by transposon mutagenesis and subcloning on a minimum DNA fragment of 6.6 kb. Broad host range plasmids containing the A. chroococcum ppc gene complemented the mutation in Fos 189 thereby restoring aerotolerant nitrogen fixation.     

Effect of Sorghum vulgare phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate carboxylase coexpression on succinate production in mutant strains of Escherichia coli

Sorghum vulgare phosphoenolpyruvate carboxylase (PEPC) and Lactococcus lactis pyruvate carboxylase (PYC) were overexpressed in Escherichia coli concurrently to improve the production of succinate, a valuable industrial specialty chemical. This coexpression system was also applied to E. coli mutant strains strategically designed by inactivating the competing pathways of succinate formation. The highest level of succinate production was observed in E. coli strains coexpressing both PEPC and PYC when compared with E. coli strains individually overexpressing either PEPC or PYC. Lactate production was also significantly reduced with PEPC and PYC coexpression. Lactate and acetate pathways were inactivated to eliminate the competing pathways of succinate formation. Results showed that inactivation of both the lactate and acetate pathways with the coexpression of PEPC and PYC was most effective in improving succinate production. Inactivating the lactate or acetate pathway alone only caused a majority of the carbon flux to shift to other metabolites rather than succinate. Coexpression of PEPC and PYC was also applied to an E. coli mutant strain deficient in lactate dehydrogenase and pyruvate:formate lyase that accumulated a substantial amount of the intermediate metabolite pyruvate during growth. Results showed that PEPC and PYC coexpression was effective in depleting pyruvate accumulation and increasing the production of metabolites.     

13C Metabolic Flux Analysis of Escherichia coli Engineered for Gamma‐Aminobutyrate Production

Escherichia coli is engineered for γ‐aminobutyrate (GABA) production in glucose minimal medium. For this, overexpression of mutant glutamate decarboxylase (GadB) and mutant glutamate/GABA antiporter (GadC), as well as deletion of GABA transaminase (GabT), are accomplished. In addition, the carbon flux to the tricarboxylic acid cycle is engineered by the overexpression of gltA, ppc, or both. The overexpression of citrate synthase (CS), encoded by gltA, increases GABA productivity, as expected. Meanwhile, the overexpression of phosphoenolpyruvate carboxylase (PPC) causes a decrease in the rate of glucose uptake, resulting in a decrease in GABA production. The phenotypes of the strains are characterized by ¹³C metabolic flux analysis (¹³C MFA). The results reveal that CS overexpression increases glycolysis and anaplerotic reaction rates, as well as the citrate synthesis rate, while PPC overexpression causes little changes in metabolic fluxes, but reduces glucose uptake rate. The engineered strain produces 1.2 g L^?1 of GABA from glucose. Thus, by using ¹³C MFA, important information is obtained for designing metabolically engineered strains for efficient GABA production.     

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation

Summary Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod^-) or nitrogen fixation (Fix^-). Seven Nod^- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod^- mutants and 14 Fix^- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His^- Nod^- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix^- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod^- mutants and one His^-Nod^- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod^- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.     

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 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.     

Strain-Specific Inhibition of nod Gene Induction in Bradyrhizobium japonicum by Flavonoid Compounds

A broad-host-range plasmid, pEA2-21, containing a Bradyrhizobium japonicum nodABC'-'lacZ translational fusion was used to identify strain-specific inhibitors of the genes required for soybean nodulation, the common nod genes. The responses of type strains of B. japonicum serogroups USDA 110, USDA 123, USDA 127, USDA 129, USDA 122, and USDA 138 to nod gene inhibitors were compared. Few compounds inhibited nod gene expression in B. japonicum USDA 110. In contrast, nod gene expression in strains belonging to several other serogroups was inhibited by most of the flavonoids tested. However, the application of two of these strain-specific compounds, chrysin and naringenin, had little effect on the pattern of competition between indigenous and inoculum strains of B. japonicum in greenhouse and field trials. Preliminary studies with radiolabeled chrysin and naringenin suggest that the different responses to nod gene inhibitors may be partly due to the degree to which plant flavonoids can be metabolized by each strain.     

Expression of an Anaplerotic Enzyme, Pyruvate Carboxylase, Improves Recombinant Protein Production in Escherichia coli

Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in Escherichia coli containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, β-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.     

DNA sequences in chromosomes 11 and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains

Summary A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the K _m values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.A preliminary report of this work was presented at the 15th International Conference on Yeast Genetics and Molecular Biology, The Hague, Netherlands. Abstract appeared in Yeast 6, S-240 (1990)     

Enzymological evidence for the function of a plastid-located pyruvate carboxylase in the Haptophyte alga Emiliania huxleyi: a novel pathway for the production of C4 compounds

Pyruvate carboxylase (PYC) catalyzes the β-carboxylation of pyruvate to yield oxaloacetate (OAA). We previously isolated a cDNA encoding a putative PYC (EhPYC1) from the haptophyte alga Emiliania huxleyi and then proposed that EhPYC1 contributes to active anaplerotic β-carboxylation during photosynthesis although PYC activity was not detected in the cell extracts. Involvement of PYC in photosynthetic carbon metabolism is unique, since PYC generally functions in non-photosynthetic organisms. In the present study, we demonstrate that EhPYC1 is highly sensitive to endogenous proteases and therefore is easily degraded in cell extracts. By avoiding proteolytic degradation, PYC activity can be detected in the cell extracts of E. huxleyi. The activity of a recombinant His-tagged EhPYC1 expressed in Streptomyces lividans was inhibited by l-malate in a mixed non-competitive manner. Immunofluorescence labeling showed that EhPYC1 is located in the plastid. This result agrees with the prediction that a bipartite plastid-targeting signal is present that functions to deliver proteins into the four-membrane plastid of haptophyte algae. This is the first finding of a plastid-located PYC. These results indicate that E. huxleyi possesses a unique pathway to produce OAA catalyzed by PYC, and the pathway may provide carbon skeletons for amino acid biosynthesis in the plastid. A database search indicates that PYC genes are widespread in green algae, diatoms and brown algae, suggesting the crucial role of PYC in various aquatic phototrophs.     

Production of phenylalanine and organic acids by phosphoenolpyruvate carboxylase-deficient mutants ofEscherichia coli

Summary Isogenic strains ofEscherichia coli were grown aerobically in minimal medium in a 2-liter airlift fermentor to determine whether appc (phosphoenolpyruvate carboxylase) mutation had the effect of directing glucose carbon into phenylalanine synthesis. Two host strains, YMC9 (ppc ⁺) and KB285 (ppc ^–) were used, either with (Phe^c) or without (Phe⁰) a plasmid which determines constitutive phenylalanine production. Carbon consumption and metabolic products were monitored. Phenylalanine production occurred only in strains carrying the Phe^c plasmid.ppc ^– strains produced less cell mass and more acetate, pyruvate, and phenylalanine (in the Phe^c strains) than did isogenicppc ⁺ strains. Lactate and ethanol production were not detected in any of the strains. Phe^c strains produced less acetate and pyruvate than their Phe⁰ homologs. Importantly,ppc ^–/Phe^c produced at least six times as much phenylalanine (0.32 g phenylalanine/g dry weight cells) asppc ⁺/Phe^c. Even in this case, however, phenylalanine was produced at ten-fold lower levels than acetate. Thus, although theppc ^– mutation stimulates phenylalanine production, it also stimulates the production of unwanted by-products such as acetate and pyruvate.     

Anaerobic fermentation of Salmonella typhimurium with and without pyruvate carboxylase

A plasmid that expressed pyruvate carboxylase (PYC) from Rhizobium etli was introduced into Salmonella typhimurium LT2. Anaerobic fermentations of S. typhimurium with and without PYC were compared with glucose as a carbon source. The presence of PYC increased the succinate yield from glucose from 0.044 g g^–1 to 0.22 g g^–1, while the lactate yield decreased from 0.31 g g^–1 to 0.16 g g^–1. Metabolic flux calculations during the early growth phase indicate that under these growth conditions in the presence of PYC more carbon flows to oxaloacetate via pyruvate carboxylase than via phosphoenolpyruvate carboxylase. Also, under these growth and induction conditions, the presence of PYC diminished the cell growth rate from 0.34 h^–1 to 0.28 h^–1, the specific rate of ATP formation from 45 mmol l^–1 h^–1 to 27 mmol l^–1 h^–1, and the specific rate of glucose consumption from 17 mmol l^–1 h^–1 to 10 mmol l^–1 h^–1.