The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression

Summary The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal c. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5 and 3 flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103154) which shows 34% similarity with the respective E. coli enzyme. Present address: Institut für Biotechnologie 1 der Kernforschungsanlage, Postfach 1913, D-5170 Jülich, Federal Republic of Germany     

A requirement for phosphoenolpyruvate carboxylase in the cyanobacterium Synechococcus PCC 7942

The gene encoding phosphoenolpyruvate carboxylase (PEPCase) in the cyanobacterium Synechococcus PCC 7942 has been isolated and characterized. As a first step in determining the role of this enzyme in cyanobacterial carbon metabolism we have attempted to generate PEPCase deficient mutants by insertional inactivation of the PEPCase gene (ppc) and recombination into the wild-type genome. Transformants generated by these constructs appear to be merodiploids in which some copies of ppc remain intact and PEPCase activity is present. Successful insertional inactivation of regions of the genome on either side of ppc suggest that the merodiploid state is a result of a requirement for PEPCase activity by the cyanobacteria. Attempts to select for ppc mutants by nutritional complementation during segregation are also described.Abbreviations PEPCase phosphenolpyruvate carboxylase - ppc gene coding for PEPCase - amp ampicillin - spec spectinomycin     

Anaplerotic Function of Phosphoenolpyruvate Carboxylase in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> USDA110

In bacteria, anaplerotic carbon fixation necessary for growth on carbon sources that are metabolized to three-carbon intermediates is provided by the activity of pyruvate carboxylase (PYC) and/or phosphoenolpyruvate carboxylase (PPC). In contrast to other rhizobia, which encode only one of these enzymes in their genomes, Bradyrhizobium japonicum USDA110 encodes both. Streptavidin-HRP western blot analysis of B. japonicum extracts demonstrated the presence of a biotin-containing protein whose molecular mass was indistinguishable from those of PYCs produced by Sinorhizobium meliloti and Rhizobium etli. Sequence analysis of the possible B. japonicum PYC revealed the lack of a pyruvate binding site as well as other characteristics indicating that the enzyme is non-functional, and PPC activity, but not PYC activity, was detectible in extracts prepared from strain USDA110. A B. japonicum cosmid genomic library was used to clone the ppc by functional complementation of S. meliloti pyc mutant RmF991. S. meliloti RmF991-carrying plasmids containing the B. japonicum ppc regained the ability to grow with glucose as a carbon source and produced PPC activity. The cloned ppc gene was inactivated by insertion mutagenesis and recombined into the USDA110 genome. The resulting ppc mutant was essentially devoid of PPC activity and grew poorly with glucose as carbon source in comparison to the wild-type strain. These data indicate that B. japonicum utilizes PPC, and not PYC, as an anaplerotic enzyme for growth on carbon sources metabolized to three-carbon intermediates.     

Metabolic characterisation of E. coli citrate synthase and phosphoenolpyruvate carboxylase mutants in aerobic cultures

E. coli is still one of the most commonly used hosts for protein production. However, when it is grown with excess glucose, acetate accumulation occurs. Elevated acetate concentrations have an inhibitory effect on growth rate and recombinant protein yield, and thus elimination of acetate formation is an important aim towards industrial production of recombinant proteins. Here we examine if over-expression of citrate synthase (gltA) or phosphoenolpyruvate carboxylase (ppc) can eliminate acetate production. Knock-out as well as over-expression mutants were constructed and characterized. Knocking out ppc or gltA decreased the maximum cell density by 14% and increased the acetate excretion by 7%, respectively decreased it by 10%. Over-expression of ppc or gltA increased the maximum cell dry weight by 91% and 23%, respectively. No acetate excretion was detected at these increased cell densities (35 and 23 g/l, respectively).     

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.     

Construction of a threonine-hyperproducing strain of Serratia marcescens by amplifying the phosphoenolpyruvate carboxylase gene

Summary The phosphoenolpyruvate carboxylase gene (ppc) of Escherichia coli K-12 was cloned on the multi-copy plasmid pLG339. Plasmid pST101, which carried a 4.3-kb SalI fragment, was introduced into Serratia marcescens T-1165, which carried the seven regulatory mutations for three aspartokinases and two homoserine dehydrogenases. Strain T-1165[pST101] produced phosphoenolpyruvate carboxylase at a rate 26 times higher than the control strain T-1165[pLG339]. While T-1165[pST101] produced 63 mg/ml l-threonine in a medium containing sucrose and urea, whereas T-1165 only produced 52 mg/ml.     

Cloning and characterization of hydrogenase genes from Azotobacter chroococcum

Summary Genomic DNA from Azotobacter chroococcum was shown by DNA hybridization to contain sequences homologous to Rhizobium japonicum H₂-uptake (hup) hydrogenase genes carried on the plasmid pHU1. Two recombinant cosmid clones, pACD101 and pACD102, were isolated from a gene library of A. chroococcum by colony hybridization and physically mapped. Each contained approximately 42 kb of insert DNA with approximately 27 kb of overlapping DNA. Further hybridization studies using three fragments from pHU1 (6 kb HindIII, 6.4 kb BglII and 5 kb EcoRI) showed that the hup-specific regions of R. japonicum and A. chroococcum are probably highly conserved. Weak homology to the hydrogenase structural genes from Desulfovibrio vulgaris (Hildenborough) was also observed. A 24 kb BamHI fragment from pACD102 subcloned into a broad host-range vector restored hydrogenase activity to several Hup^- mutants of A. chroococcum.     

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     

基于基因组的一株土壤固氮菌分离菌株鉴定及其促生作用

[目的] 为获得高效固氮菌株,充分研究利用土壤固氮菌资源。[方法] 选取固氮能力较高的紫色土发育水稻土,采用富集纯化法分离固氮微生物菌株。通过16S rRNA基因系统发育分析和全基因组相关指数比较对新分离菌株进行物种鉴定。采用乙炔还原法和¹⁵N₂示踪法定量测定新分离菌株的固氮能力,通过培养特性和接种效果初步研究固氮菌株的促生作用。[结果] 从紫色土发育水稻土中分离得到1株可在无氮培养基上快速生长的菌株P208。基于16S rRNA基因和基因组92个核心基因的系统发育分析结果表明,新分离菌株P208与Azotobacter chroococcum IAM 12666^T（=ATCC 9043^T）系统发育距离最近（16S rRNA基因相似度为99.79%）。菌株P208与A.chroococcum ATCC 9043^T的基因组平均核苷酸一致性（ANI）、平均氨基酸一致性（AAI）和数字DNA-DNA杂交值（dDDH）高于物种分类阈值（ANI>95%-96%,AAI>95%-96%,dDDH>70%）,最大唯一匹配指数（MUMi）低于物种分类阈值（<0.33）,得出新分离菌株P208为褐球固氮菌（A.chroococcum）。A.chroococcum P208固氮活性为模式菌株A.chroococcum ATCC 9043^T的2.61倍。除固氮能力外,A.chroococcum P208具有IAA生成、溶磷活性和铁载体生成等促进植物生长潜力的培养特性,室内培养条件下接种A.chroococcum P208能够促进水稻、小麦幼苗根系的生长。[结论] 从固氮能力较强的水稻土中分离纯化得到1株具有较强固氮、促生潜力的固氮菌,具有潜在的开发应用价值,可为研究利用生物固氮提供微生物资源。     

Effect of Overexpression of Actinobacillus succinogenes Phosphoenolpyruvate Carboxykinase on Succinate Production in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold.     

Phosphoenolpyruvate carboxylase in soybean root nodules: An immunochemical study

The activity of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) strongly increased during the maturation of soybean (Glycine max L. Weber) root-nodules. By using a specific immune serum it was shown that this increase was the consequence of an elevated population of enzyme molecules whose appearance preceded the emergence of nitrogen fixing capacity. Whether or not the phenomenon could be ascribed to the formation of a specific isoenzyme is not known. The location of the enzyme was also investigated. Immunocyto-fluorescence experiments established that phosphoenolpyruvate carboxylase was present in the cytoplasmic compartment of both infected and uninfected cells of nodules.Abbreviation PEPCase phosphoenolpyruvate carboxylase     

The nifH, nifM and nifN genes of Azotobacter vinelandii: Characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks

Summary Tn5 was introduced into Azotobacter vinelandii on a suicide vector, pGS9. Three Nif^- mutants were found to carry Tn5 in nifH (MV6), in nifN (MV22), and in or near nifM (MV21), from the results of hybridisation experiments. For MV21 and MV22 this was also shown by complementation with the nif genes of Klebsiella pneumoniae on pRD1. MV6 failed to synthesis the nifH, D and K gene products. MV6 and MV22 fixed nitrogen in the absence of supplied molybdenum while mutant MV21 did not, suggesting that the nifM gene product may be required for the alternative nitrogenase system synthesised in azotobacteria under conditions of molybdenum deprivation. Reconstitution experiments with mutant extracts showed that MV22 (nifN ^-) lacked the FeMo cofactor and that MV21 (NifM^-) synthesised inactive Fe protein. These biochemical phenotypes are identical to those of the K. pneumoniae nifN and nifM mutants, respectively, demonstrating that these genes have the same function in both K. pneumoniae and A. vinelandii. Complementation of the A. vinelandii mutants with pLAFR1 gene banks of A. vinelandii or a. chroococcum yielded three cosmids of interest. pLV10 complemented UW91, a nifH mutant, and corrected the defect in MV6 after recombination with the mutant genome. It also carried nifD (but not nifK) and about 18 kb of DNA upstream from nifH. pLV1 from the A. vinelandii gene bank complemented both MV21 and MV22 as did pLC11, isolated from the A. chroococcum gene bank. Both pLV1 and pLC11 carried part of the nif cluster downstream of nifHDK which also includes nifEN and nifMVS on about 22 kb of DNA.     

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.     

Studies on the central metabolism of Thermus aquaticus,an extreme thermophilic bacterium: Anaplerotic reactions and their regulation

The physiology of Thermus aquaticus strain Z05 was investigated. Substantial evidence for gene and enzyme regulation in the central metabolism of this extreme thermophile was found.Two anaplerotic pathways were detected: (1) phosphoenolpyruvate carboxylase; (2) a glyoxylate shunt which proved to be essential for growth on pyruvate as well as acetate. The synthesis of isocitrate lyase and malate synthase were found to depend on a common control mechanism. Pronounced regulatory effects were observed on the activity of malic enzyme, pyruvate kinase and phosphoenolpyruvate carboxylase. The data could be fitted together into a picture of the metabolism during glycolysis and gluconeogenesis which shows how variations of enzyme levels and activities correlate with the apparent needs of the cell.Our results call attention to a peculiar metabolic analogy between T. aquaticus and Acinetobacter Abbreviations ace acetate nonutilizing - Acetyl-CoA acetyl-coenzyme A - I.U. international unit - PEP phosphoenolpyruvate - T Thermus     

Metabolic engineering of Propionibacterium freudenreichii: effect of expressing phosphoenolpyruvate carboxylase on propionic acid production

Propionic acid is currently produced mainly via petrochemicals, but there is increasing interest in its fermentative production from renewable biomass. However, the current propionic acid fermentation process suffers from low product yield and productivity. In this work, the gene encoding phosphoenolpyruvate carboxylase (PPC) was cloned from Escherichia coli and expressed in Propionibacterium freudenreichii. PPC catalyzes the conversion of phosphoenolpyruvate to oxaloacetate with the fixation of one CO₂. Its expression in P. freudenreichii showed profound effects on propionic acid fermentation. Compared to the wild type, the mutant expressing the ppc gene grew significantly faster, consumed more glycerol, and produced propionate to a higher final titer at a faster rate. The mutant also produced significantly more propionate from glucose under elevated CO₂ partial pressure. These effects could be attributed to increased CO₂ fixation and resulting changes in the flux distributions in the dicarboxylic acid pathway.     

Nitrogen fixation in strains ofAzotobacter chroococcum bearing deletions of a cluster of genes coding for nitrogenase

Strains of the obligately aerobic nitrogen fixing organismAzotobacter chroococcum were constructed which contained defined chromosomal deletions in which the nitrogenase structural genenifHDK cluster (nifH for the polypeptide of the Fe-protein component of nitrogenase andnifD andnifK for the alpha and beta subunits respectively of the MoFe-protein component of the enzyme) was replaced by a kanamycin resistance gene. N₂ fixation was nevertheless observed in deletion strains though only in a molybdenum-deficient medium or in spontaneously arising tungstate-resistant derivatives. In comparison with the parent strain growing in molybdenum-sufficient medium, diazotrophic growth was slow and the nitrogenase activity in vivo was characterised by disproportionately low rates of C₂H₂-reduction compared to H₂-evolution and relative insensitivity of H₂-evolution to inhibition by C₂H₂. The findings show reiteration of functional structural genes for nitrogenase inA. chroococcum consistent with our previous observation of twonifH genes in this organism and detection in this work of a secondnifK-like sequence in the genomes of both parent and deletion strains whenA. chroococcum nifK DNA was used as a probe.     

Hydrogen-stimulated CO2 fixation and coordinate induction of hydrogenase and ribulosebiphosphate carboxylase in a H2-uptake positive strain of Rhizobium japonicum

H₂-uptake positive strains (122 DES and SR) and H₂-uptake negative strains SR2 and SR3 of Rhizobium japonicum were examined for ribulosebisphosphate (RuBP) carboxylase and H₂-uptake activities during growth conditions which induced formation of the hydrogenase system. The rate of ¹⁴CO₂ uptake by hydrogenase-derepressed cells was about 6-times greater in the presence than in the absence of H₂. RuBP carboxylase activity was observed in free-living R. japonicum strains 122 DES or SR only when the cells were derepressed for their hydrogenase system. Hydrogenase and RuBP carboxylase activities were coordinately induced by H₂ and both were repressed by added succinate. Hydrogenase-negative mutant strains SR2 and SR3 derived from R. japonicum SR showed no detecyable RuBP carboxylase activities under hydrogenase derepression conditions. No detectable RuBP carboxylase was observed in bacteroids formed by H₂-uptake positive strains R. japonicum 122 DES or SR. Propionyl CoA carboxylase activity was consistently observed in extracts of cells from free-living cultures of R. japonicum but activity was not appreciably influenced by the addition of H₂. Neither phosphoenolpyruvate carboxylase nor phosphoenolpyruvate carboxykinase activity was detected in extracts of R. japonicum.Abbreviations RuBP Ribulose 1,5-bisphosphate - (Na₂EDTA) (Ethylenedinitrilo)-tetraacetic acid, disodium salt - (propionyl CoA) Propionyl coenzyme A - (PEP) Phosphoenolpyruvate - (GSH) Reduced glutathione - (Tricine) N-tris(hydroxymethyl)-methylglycine     

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 mol·(mg protein)^-1·min^-1 and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. K_m values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (K_a) for Mg²⁺ was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.Abbreviations MW molecular weight - PEP (Case) phosphoenolpyruvate (carboxylase) - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis