Cloning and characterization of the gene encoding phosphoketolase in <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> isolated from kimchi

The gene encoding phosphoketolase, which is 2749 bp long and contains 814 amino acid polypeptides with a total molecular mass of 91.9 kDa, was cloned from Leuconostoc mesenteroides C7 (LMC7) and expressed in Escherichia coli. It exhibited a homology of >58% with phosphoketolases from other lactic acid bacteria. The phosphoketolase of LMC7 belongs to the xylulose 5-phosphate (X5P)/fructose 6-phosphate (F6P) phosphoketolase (Xfp) family, which is an enzyme with dual specificity for X5P and F6P. The members of this family contain typical thiamin pyrophosphate (TPP) binding sites as reported for other TPP-dependent enzymes, and several highly conserved regions as signature patterns for phosphoketolases. The plasmid pGPK containing the Xfp gene (xfp) exhibits phosphoketolase activity in E. coli. The specific activities of the enzyme from E. coli BL21 and E. coli EC101 harboring xfp were 0.28 and 0.14 units/mg, respectively. They both exhibited a 1.5-fold increase in the production of acetic acid from acetyl phosphate compared with their corresponding original strain.An erratum to this article can be found at     

Physiological conditions conducive to high cell density and high cyanophycin content in <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> strain H16 possessing a KDPG aldolase gene-dependent addiction system

The recombinant strain of Ralstonia eutropha H16-PHB⁻4-∆eda (pBBR1MCS-2::cphA ₆₃₀₈/eda _H16) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH₃, K₂SO₄, MgSO₄⋅7H₂O, Fe(Ш)NH₄-citrate, CaCl₂⋅2H₂O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbA_Re) and NADPH-dependent acetoacetyl-CoA reductase (PhbB_Re) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1 _Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB⁻4 to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1 _Ps gene only. In addition, recombinant strains of R. eutropha PHB⁻4 produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB⁻4/pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units. Received: 12 July 1999 / Received revision: 1 October 1999 / Accepted: 2 October 1999     

Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC₅₀) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen Cryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.     

Production of polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from an oil remaining from biotechnological rhamnose production

Screening experiments identified several bacteria which were able to use residual oil from biotechnological rhamnose production as a carbon source for growth. Ralstonia eutropha H16 and Pseudomonas oleovorans were able to use this waste material as the sole carbon source for growth and for the accumulation of polyhydroxyalkanoic acids (PHA). R. eutropha and P. oleovorans accumulated PHA amounting to 41.3% and 38.9%, respectively, of the cell dry mass, when these strains were cultivated in mineral salt medium with the oil from the rhamnose production as the sole carbon source. The accumulated PHA isolated from R. eutropha consisted of only 3-hydroxybutyric acid, whereas the PHA isolated from P. oleovorans consisted of 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxy decanoic acid, and 3-hydroxydodecanoic acid. The composition was confirmed by gas chromatography of the isolated polyesters. Batch and fed-batch cultivations in stirred-tank reactors were done. Received: 15 June 1999 / Received revision: 10 August 1999 / Accepted: 13 August 1999     

Different glycolytic pathways for glucose and fructose in the halophilic archaeon Halococcus saccharolyticus

The glucose and fructose degradation pathways were analyzed in the halophilic archaeon Halococcus saccharolyticus by ¹³C-NMR labeling studies in growing cultures, comparative enzyme measurements and cell suspension experiments. H. saccharolyticus grown on complex media containing glucose or fructose specifically ¹³C-labeled at C1 and C3, formed acetate and small amounts of lactate. The ¹³C-labeling patterns, analyzed by ¹H- and ¹³C-NMR, indicated that glucose was degraded via an Entner-Doudoroff (ED) type pathway (100%), whereas fructose was degraded almost completely via an Embden-Meyerhof (EM) type pathway (96%) and only to a small extent (4%) via an ED pathway. Glucose-grown and fructose-grown cells contained all the enzyme activities of the modified versions of the ED and EM pathways recently proposed for halophilic archaea. Glucose-grown cells showed increased activities of the ED enzymes gluconate dehydratase and 2-keto-3-deoxy-gluconate kinase, whereas fructose-grown cells contained higher activities of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase. During growth of H. saccharolyticus on media containing both glucose and fructose, diauxic growth kinetics were observed. After complete consumption of glucose, fructose was degraded after a lag phase, in which fructose-1-phosphate kinase activity increased. Suspensions of glucose-grown cells consumed initially only glucose rather than fructose, those of fructose-grown cells degraded fructose rather than glucose. Upon longer incubation times, glucose- and fructose-grown cells also metabolized the alternate hexoses. The data indicate that, in the archaeon H. saccharolyticus, the isomeric hexoses glucose and fructose are degraded via inducible, functionally separated glycolytic pathways: glucose via a modified ED pathway, and fructose via a modified EM pathway.Abbreviations. KDG 2-Keto-3-deoxygluconate - KDPG 2-Keto-3-deoxy-6-phosphogluconate - FBP Fructose-1,6-bisphosphate - TIM Triosephosphate isomerase - GAP Glyceraldehyde-3-phosphate - PEP Phosphoenolpyruvate - PTS Phosphotransferase - 1-PFK Fructose 1-phosphate kinase An erratum to this article can be found at     

HETEROLOGOUS EXPRESSION OF Escherichia coli FRUCTOSE-1,6-BISPHOSPHATASE IN Corynebacterium glutamicum AND EVALUATING THE EFFECT ON CELL GROWTH AND L-LYSINE PRODUCTION

Fructose-1,6-bisphosphatase (FBPase), which is mainly used to supply NADPH, has an important role in increasing L-lysine production by Corynebacterium glutamicum. However, C. glutamicum FBPase is negatively regulated at the metabolic level. Strains that overexpressed Escherichia coli fructose-1,6-bisphosphatase in C. glutamicum were constructed, and the effects of heterologous FBPase on cell growth and L-lysine production during growth on glucose, fructose, and sucrose were evaluated. The heterologous fructose-1,6-bisphosphatase is insensitive to fructose 1-phosphate and fructose 2,6-bisphosphate, whereas the homologous fructose-1,6-bisphosphatase is inhibited by fructose 1-phosphate and fructose 2,6-bisphosphate. The relative enzyme activity of heterologous fructose-1,6-bisphosphatase is 90.8% and 89.1% during supplement with 3 mM fructose 1-phosphate and fructose 2,6-bisphosphate, respectively. Phosphoenolpyruvate is an activator of heterologous fructose-1,6-bisphosphatase, whereas the homologous fructose-1,6-bisphosphatase is very sensitive to phosphoenolpyruvate. Overexpression of the heterologous fbp in wild-type C. glutamicum has no effect on L-lysine production, but fructose-1,6-bisphosphatase activities are increased 9- to 13-fold. Overexpression of the heterologous fructose-1,6-bisphosphatase increases L-lysine production in C. glutamicum lysC ^T311I by 57.3% on fructose, 48.7% on sucrose, and 43% on glucose. The dry cell weight (DCW) and maximal specific growth rate (μ) are increased by overexpression of heterologous fbp. A “funnel-cask” diagram is first proposed to explain the synergy between precursors supply and NADPH supply. These results lay a definite theoretical foundation for breeding high L-lysine producers via molecular target.     

Physiological–biochemical properties and the ability to synthesize polyhydroxyalkanoates of the glucose-utilizing strain of the hydrogen bacterium Ralstonia eutropha B8562

Physiological–biochemical, genetic, and cultural properties of the glucose-utilizing mutant strain Ralstonia eutropha B8562 have been compared with those of its parent strain R. eutropha B5786. It has been shown that growth characteristics of the strain cultured on glucose as the sole carbon and energy source are comparable with those of the parent strain. Strain B8562 is characterized by high polyhydroxyalkanoate (PHA) yields on different carbon sources (CO₂, fructose, and glucose). PHA accumulation in the strain batch cultured on glucose under nitrogen deficiency reaches 90 %. The major monomer in the PHA is β-hydroxybutyric acid (more than 99 mol %); the identified minor components are β-hydroxyvaleric acid (0.25–0.72 mol %) and β-hydroxyhexanoic acid (0.08–1.5 mol %). The strain is a promising PHA producer on available sugar-containing media with glucose.     

Synthesis and Some Properties of UDP-(1)fructose

UDP-(1)fructose was synthesized essentially by the method of Michelson or Roseman et al. The product obtained was much more stable to acid than UDP-fructose isolated from Jerusalem artichoke tubers by Umemura et al.⁷⁾ and UDP-glucose. Hydrolysis time curves of UDP-(1)fructose and fructose-1-phosphate in 0.01N HGl and 0.1N HCl both at 100°C are presented. It was concluded from these curves that UDP-(1)fructose was first hydrolyzed into UMP and fructose-1-phosphate, and then fructose-1-phosphate was hydrolyzed more slowly into free fructose and inorganic phosphate.     

Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity

Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6-P₂) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P₂ from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa.Abbreviations ALD Fru-1,6-P₂ aldolse - DHAP dihydroxyacetone phosphate - F6P fructose 6-phosphate - G6P glucose 6-phosphate - Gly3P glyceraldehyde 3-phosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - PFK fructose 6-phosphate kinase - PGI phosphoglucose isomerase - 6PG 6-phosphogluconate     

Fructose-1,6-bisphosphatase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>: expression and deletion of the<Emphasis Type="Italic"> fbp</Emphasis> gene and biochemical characterization of the enzyme

The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K_m value of about 14 µM and a V_max of about 5.4 µmol min^–1 mg^–1 and k_cat of about 3.2 s^–1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg²⁺ or Mn²⁺ and was inhibited by the monovalent cation Li⁺ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.     

A lower specificity PhaC2 synthase from Pseudomonas stutzeri catalyses the production of copolyesters consisting of short-chain-length and medium-chain-length 3-hydroxyalkanoates

A polyhydroxyalkanoate (PHA) synthase gene phaC2 _Ps from Pseudomonas stutzeri strain 1317 was introduced into a PHA synthase gene phbC _Re negative mutant, Ralstonia eutropha PHB⁻4. It conferred on the host strain the ability to synthesize PHA, the monomer compositions of which varied widely when grown on different carbon sources. During cultivation on gluconate, the presence of phaC2 _Ps in R. eutropha PHB⁻4 led to the accumulation of polyhydroxybutyrate (PHB) homopolymer in an amount of 40.9 wt% in dry cells. With fatty acids, the recombinant successfully produced PHA copolyesters containing both short-chain-length and medium-chain-length 3-hydroxyalkanoate (3HA) of 4–12 carbon atoms in length. When cultivated on a mixture of gluconate and fatty acid, the monomer composition of accumulated PHA was greatly affected and the monomer content was easily regulated by the addition of fatty acids in the cultivation medium. After the (R)-3-hydroxydecanol-ACP:CoA transacylase gene phaG _Pp from Pseudomonas putida was introduced into phaC2 _Ps-containing R. eutropha PHB⁻4, poly(3HB-co-3HA) copolyester with a very high 3-hydroxybutyrate (3HB) fraction (97.3 mol%) was produced from gluconate and the monomer compositions of PHA synthesized from fatty acids were also altered. This study clearly demonstrated that PhaC2_Ps cloned from P. stutzeri 1317 has extraordinarily low substrate specificity in vivo, though it has only 54% identity in comparison to a previously described low-substrate-specificity PHA synthase PhaC1_Ps from Pseudomonas sp. 61–3. This study also indicated that the monomer composition and content of the synthesized PHA can be effectively modulated by controlling the addition of carbon sources or by modifying metabolic pathways in the hosts.     

Der Biosyntheseweg der RNS-Ribose in Hydrogenomonas eutropha Stamm H 16 und Pseudomonas facilis

Zusammenfassung Die am Fructose- und Gluconatabbau über den Entner-Doudoroff-Weg beteiligten Enzyme sowie die Enzyme des oxydativen Pentosephosphat-Weges wurden in Rohextrakten von Hydrogenomonas eutropha Stamm H 16 und Pseudomonas facilis, sowohl nach autotrophem Wachstum als auch nach heterotrophem Wachstum auf Fructose oder Gluconat, bestimmt. Fructose induziert in H. eutropha alle Enzyme des Entner-Doudoroff-Weges, Gluconat nur die Gluconokinase, die 6-Phosphogluconat-Dehydratase und die 2-Keto-3-desoxy-6-phosphogluconat-Aldolase. Dagegen induzieren in P. facilis beide Substrate den gesamten Enzymsatz. Das Fehlen der 6-Phosphogluconat-Dehydrogenase in H. eutropha und das Vorhandensein einer NAD-abhängigen 6-Phosphogluconat-Dehydrogenase in P. facilis wurden bestätigt. Die Enzymaktivitäten in voll induzierten, auf Fructose gewachsenen Zellen beider Arten sind ähnlich.Mit beiden Stämmen wurden Einbauexperimente mit U-¹⁴C-, 1-¹⁴C- und 6-¹⁴C-Fructose sowie 1-¹⁴C- und 6-¹⁴C-Gluconat als Substrate durchgeführt. Die Ribose wurde aus der RNS isoliert und durch Lactobacillus plantarum fermentativ in Essigund Milchsäure gespalten. Die spezifische Radioaktivität der einzelnen C-Atome wurde durch schrittweisen Abbau der Säuren, quantitative Bestimmung des dabei entstehenden ¹⁴CO₂ und Messung der darin enthaltenen absoluten Radioaktivität ermittelt.Die Ergebnisse zeigen, daß die Ribose in Stamm H 16 ausschließlich über die nicht-oxydativen Reaktionen des Pentosephosphat-Weges gebildet wird. Die C-Atome 1,2 und 3 des Gluconats tragen nicht signifikant zur Gluconeogenese bei.Das Markierungsmuster der Ribose aus P. facilis ist mit dem von Stamm H 16 nahezu identisch. Die oxydativen Reaktionen des Pentosephosphat-Weges über die 6-Phosphogluconat-Dehydrogenase sind von quantitativ geringerer Bedeutung als die Transaldolase-Transketolase-Reaktionen.

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The biosynthetic pathway of RNA ribose in Hydrogenomonas eutropha Strain H 16 and Pseudomonas facilis

Summary The enzymes involved in the degradation of fructose and gluconate via the Entner-Doudoroff-pathway as well as those involved in the oxidative pentose phosphate pathway have been determined in crude extracts of Hydrogenomonas eutropha strain H 16 and of Pseudomonas facilis after either autotrophic growth or heterotrophic growth on fructose or gluconate as substrates. In H. eutropha fructose induces all enzymes of the Entner-Doudoroff-pathway, gluconate induces only glucokinase, 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase. In contrast, in P. facilis both substrates induce the entire set of enzymes. The absence of 6-phosphogluconate dehydrogenase in H. eutropha and the presence of a NAD-linked 6-phosphogluconate dehydrogenase in P. facilis have been confirmed. Otherwise, the enzyme activities in fully induced fructose grown cells of both species are similar.Incorporation experiments were performed using both bacterial species and employing U-¹⁴C-, 1-¹⁴C-, and 6-¹⁴C-fructose as well as 1-¹⁴C- and 6-¹⁴C-gluconate as substrates. Ribose was isolated from RNA and fermented by Lactobacillus plantarum with the production of acetic and lactic acids. By stepwise degradation of the acids and by quantitative measurement and scintillation counting of the carbon dioxide formed the specific radioactivity of each carbon atom has been determined.The results demonstrate that in strain H 16 ribose is formed exclusively via the non-oxidative reactions of the pentose phosphate pathway. Carbon atoms 1 to 3 of gluconate do not significantly contribute to gluconeogenesis.With P. facilis an almost identical labelling pattern was observed, indicating that the oxidative reactions of the pentose phosphate pathway via 6-phosphogluconate dehydrogenase are quantitatively of minor importance for ribose synthesis than the transaldolase-transketolase reactions.

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Abkürzungen ATP Adenosin-5-triphosphat - DAP Dihydroxyacetonphosphat - E-4-P Erythrose-4-phosphat - ED Entner-Doudoroff - EDTA Äthylen-diamin-tetraessigsäure - FDP(ase) Fructose-1,6-diphosphat(ase) - F-6-P Fructose-6-phosphat - G-6-P(-DH) Glucose-6-phosphat(-Dehydrogenase) - GAP Glycerinaldehyd-3-phosphat - GDH Glycerin-1-phosphat-Dehydrogenase - GK Gluconokinase - HK Hexokinase - KDPG 2-Keto-3-desoxy-6-phosphogluconat - LDH Lactat-Dehydrogenase - NAD(H₂) Nicotin-amid-adenin-dinucleotid (reduziert) - NADP(H₂) Nicotinamid-adenin-dinucleotidphosphat (reduziert) - PGI Phosphoglucose-Isomerase - PP Pentosephosphat - 6-PG(-DH) 6-Phosphogluconat(-Dehydrogenase) - 6-PG-DHT 6-Phosphogluconat-Dehydratase - R-5-P Ribose-5-phosphat - Ru-5-P Ribulose-5-phosphat - Su-7-P Seduheptulose-7-phosphat - TA Transaldolase - TEA Triäthanolaminhydrochlorid - TIM Triosephosphat-Isomerase - TK Transketolase - TPP Thiaminpyrophosphat - Tris Tris-(hydroxymethyl)-aminomethan - Xu-5-P Xylulose-5-phosphat     

Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

Recombinant Ralstonia eutropha strain PHB⁻4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1_Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB⁻4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB⁻4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.     

Crystal Structures of Phosphoketolase: THIAMINE DIPHOSPHATE-DEPENDENT DEHYDRATION MECHANISM*

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H₂O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,β-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

Denitrifikation bei Hydrogenomonas eutropha Stamm H16

Zusammenfassung Hydrogenomonas eutropha (syn. Alcaligenes eutrophus) Stamm H 16 wächst anaerob mit Fructose und Nitrat bzw. Nitrit. Autotrophanaerobes Wachstum unter einer H₂-CO₂-Atmosphäre (90+10 Vol.-%) mit Nitrat als einzigem Wasserstoff-Acceptor ist minimal.Während des anaeroben Wachstums mit Nitrat sind zwei Phasen zu unterscheiden. In der ersten Phase erfolgt die Zellvermehrung auf Kosten der Reduktion von Nitrat zu Nitrit; dieses wird angehäuft. In der zweiten Phase wird Nitrit unter Bildung von Stickstoff reduziert.Gewaschene, anaerob gewachsene Zellen reduzieren Nitrat und Nitrit unter Bildung von N₂. Stöchiometrische Experimente mit H₂ oder Fructose als H-Donatoren lassen darauf schließen, daß Stickstoff das einzige Produkt der Denitrifikation durch die Zellen ist. Diese Schlußfolgerung wurde durch eine massenspektrometrische Analyse des gebildeten Gases bestätigt. Aerob gewachsene Zellen reduzieren Nitrat nur zu Nitrit. In Gegenwart von Ammonium-Salz gewachsene Zellen reduzieren Nitrat mit sehr geringer Rate.Die Ergebnisse deuten darauf hin, daß Stamm H 16 über nur eine Nitratreductase verfügt. Die Bildung des Enzyms ist durch Ammonium reprimierbar; O₂ ist ohne Einfluß. Die Nitritreductase fand sich sowohl in der löslichen Fraktion als auch in den gereinigten Partikeln lokalisiert. Das Nitritreductase-System wird nur unter anaeroben Bedingungen gebildet.

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Denitrification in Hydrogenomonas eutropha strain H16

Summary The hydrogen bacterium Hydrogenomonas eutropha (syn. Alcaligenes eutrophus) strain H 16 is able to grow anaerobically with fructose and nitrate or nitrite, respectively. Autotrophic anaerobic growth under a gas atmosphere of hydrogen and carbon dioxide (90+10 vol-%) with nitrate as the sole hydrogen acceptor is minimal.During anaerobic growth with nitrate as H-acceptor, two growth phases are distinguishable: During the first phase cell growth occurs with the reduction of nitrate to nitrite, which is accumulated; on the second phase nitrite is reduced with the formation of gaseous nitrogen.Washed, anaerobically grown cells reduce nitrate and nitrite with the formation of N₂. Stoichiometric experiments employing hydrogen or fructose as the hydrogen donors are consistent with the conclusion that nitrogen is the sole product of denitrification by these cells. This was confirmed by mass spectrometric analysis of the gas formed. Aerobically grown cells are able to reduce nitrate only to nitrite; when grown in the presence of ammonia, the reduction rate is very low.The results indicate that strain H 16 contains only one nitrate reductase. The formation of this enzyme system is not influenced by oxygen, however, is repressed by ammonia.When employing a purified soluble fraction and particles, nitrite reductases were found in both fractions. The nitrite reductase system is formed only under anaerobic conditions.

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Abkürzungen MB Methylenblau - PMS Phenazinmethosulfat     

Characteristics of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production byRalstonia eutropha NCIMB 11599 and ATCC 17699

Ralstonia eutropha NCIMB 11599 and ATCC 17699 were grown, and their productions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] compared. In flask cultures ofR. eutropha NCIMB 11599, cell concentration, P(3HB-co-4HB) concentration and polymer content decreased considerably with increases in the γ-butyrolactone concentration, and the 4HB fraction was also very low (maximum 1.74 mol%). In fed-batch cultures ofR. eutropha NCIMB 11599, glucose and γ-butyrolactone were fed as the carbon sources, under a phosphate limitation strategy. When glucose was fed as the sole carbon source, with its concentration controlled using an on-line glucose analyzer, 86% of the P(3HB) homopolymer was obtained from 201 g/L of cells. In a two-stage fed-batch culture, where the cell concentration was increased to 104 g/L, with glucose fed in the first step and constant feeding of γ-butyrolactone, at 6 g/h, in the second, final cell concentration at 67 h was 106 g/L, with a polymer content of 82%, while the 4HB fraction was only 0.7 mol%. When the same feeding strategy was applied to the fedbatch culture ofR. eutropha ATCC 17699, where the cell concentration was increased to 42 g/L, by feeding fructose in the first step and γ-butyrolactone (1.5 g/h) in the second, the final cell concentration, polymer content and 4HB fraction at 74 h were 51 g/L, 35% and 32 mol%, respectively. In summary,R. eutropha ATCC 17699 was better thanR. eutropha NCIMB 11599 in terms of P(3HB-co-4HB) production with various 4HB fractions.     

Cloning, expression, purification, cofactor requirements, and steady state kinetics of phosphoketolase-2 from Lactobacillus plantarum

The genes xpk1 and xpk2(Δ1–21) encoding phosphoketolase-1 and (Δ1–7)-truncated phosphoketolase-2 have been cloned from Lactobacillus plantarum and expressed in Escherichia coli. Both gene-products display phosphoketolase activity on fructose-6-phosphate in extracts. A N-terminal His-tag construct of xpk2(Δ1–21) was also expressed in E. coli and produced active His-tagged (Δ1–7)-truncated phosphoketolase-2 (hereafter phosphoketolase-2). Phosphoketolase-2 is activated by thiamine pyrophosphate (TPP) and the divalent metal ions Mg²⁺, Mn²⁺, or Ca²⁺. Kinetic analysis and data from the literature indicate the activators are MgTPP, MnTPP, or CaTPP, and these species activate by an ordered equilibrium binding pathway, with Me²⁺TPP binding first and then fructose-6-phosphate. Phosphoketolase-2 accepts either fructose-6-phosphate or xylulose-5-phosphate as substrates, together with inorganic phosphate, to produce acetyl phosphate and either erythrose-4-phosphate or glyceraldehyde-3-phosphate, respectively. Steady state kinetic analysis of acetyl phosphate formation with either substrate indicates a ping pong kinetic mechanism. Product inhibition patterns with erythrose-4-phosphate indicate that an intermediate in the ping pong mechanism is formed irreversibly. Background mechanistic information indicates that this intermediate is 2-acetyl-TPP. The irreversibility of 2-acetyl-TPP formation might explain the overall irreversibility of the reaction of phosphoketolase-2.     

Detection of Bifidobacteriumspecies by enzymatic methods and antimicrobial susceptibility testing

Twenty four strains of bifidobacteria and 16 strains of lactobacilli were examined for activities of fructose-6-phosphate phosphoketolase, a-galactosidase, and a-glucosidase and the resistance to metronidazole and mupirocin over 24 h. The detection of a-galactosidase, a-glucosidase, and metronidazole susceptibility gave unclear results. The mupirocin susceptibility using disc diffusion method (200 mg mupirocin per disc) had the same validity as the detection of fructose-6-phosphate phosphoketolase – the key enzyme of the bifidobacteria carbohydrate metabolism. While 24 bifidobacteria strains tested were mupirocin-resistant, 16 lactobacilli strains were susceptible. Hence, the resistance to mupirocin may serve as a simple criterion distinquishing Bifidobacterium spp. from Lactobacillus spp.