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1.
para-Aminobenzoate (PABA), a valuable chemical raw material, can be synthesized by most microorganisms. This aromatic compound is currently manufactured from petroleum-derived materials by chemical synthesis. To produce PABA from renewable resources, its production by fermentation was investigated. The evaluation of the sensitivity to PABA toxicity revealed that Corynebacterium glutamicum had better tolerance to PABA than several other microorganisms. To produce PABA from glucose, genetically engineered C. glutamicum was constructed by introducing both pabAB and pabC. The generated strain produced 20 mM of PABA in a test-tube scale culture; however, during the investigation, an unidentified major byproduct was detected in the culture supernatant. Unexpectedly, the byproduct was also detected after the incubation of PABA with glucose in a buffer solution without bacterial cells. To elucidate the mechanism underlying the formation of this byproduct, PABA analogues and several kinds of sugars were mixed and analyzed. New chemical compounds were detected when incubating aniline with glucose as well as PABA with reducing sugars (mannose, xylose, or arabinose), indicating that an amino group of PABA reacted non-enzymatically with an aldehyde group of glucose. The molecular mass of the byproduct determined by LC-MS suggested that the molecule was generated from PABA and glucose with releasing a water molecule, generally known as a glycation product. Because the glycation reaction was reversible, the byproduct was easily converted to PABA by acid treatment (around pH 2-3) with HCl. Then, pab genes were screened to improve PABA production. The highest PABA concentration was achieved by a strain expressing the pabAB of Corynebacterium callunae and a strain expressing the pabC of Xenorhabdus bovienii, respectively. A plasmid harboring both the pabAB of C. callunae and the pabC of X. bovienii, the best gene combination, was introduced into a strain overexpressing the genes of the shikimate pathway. The resultant strain produced 45 mM of PABA in a test-tube scale culture. Under a fermenter-controlled condition, the strain produced up to 314 mM (43 g/L) of PABA at 48 h, with a 20% yield. To our knowledge, this is the highest concentration of PABA produced by a genetically modified microorganism ever reported.  相似文献   

2.
Albicidins are potent DNA-gyrase-inhibiting antibiotics and phytotoxins synthesised by Xanthomonas albilineans. Functions have been deduced for some clustered biosynthetic genes, including a PKS-NRPS megasynthase, methyltransferases and regulatory genes, and resistance genes including a transporter and a gyrase-binding protein. More puzzling is the presence in this cluster of apparent aromatic metabolism genes. Here, we describe functional analysis of several such genes and propose a model for their role. An apparent benzoate CoA ligase (xabE) proved essential for albicidin production and pathogenicity. A neighbouring operon includes genes for p-aminobenzoate (PABA) metabolism. A PABA synthase fusion (pabAB) restored prototrophy in pabA and pabB mutants of Escherichia coli, proving functionality. Inactivation of pabAB increased susceptibility to sulphanilamide but did not block albicidin production. X. albilineans contains a remote pabB gene which evidently supplies enough PABA for albicidin biosynthesis in culture. Additional capacity from pabAB may be advantageous in more demanding environments such as infected plants. Downstream from pabAB are a known resistance gene (albG) and ubiC which encodes a p-hydroxybenzoate (PHBA) synthase. PHBA protects X. albilineans from inhibition by PABA. Therefore, coordinated expression may protect X. albilineans against toxicity of both the PABA intermediate and the albicidin product, under conditions that induce high-level antibiotic biosynthesis.  相似文献   

3.
We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and β-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF fbr and tyrA fbr or aroF fbr and pheA fbr ) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.  相似文献   

4.
Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving l-phenylalanine (l-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of l-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of l-Phe. In contrast, overexpression of aroF wt or pheA fbr from E. coli significantly increased l-Phe production. Co-overexpression of aroF wt and pheA fbr improved the titer of l-Phe to 4.46 ± 0.06 g l?1. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of l-Phe production. Co-overexpression of the mutated pheA fbr and the wild-type gene aroH resulted in the production of l-Phe up to 4.64 ± 0.09 g l?1. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.  相似文献   

5.
Shikimic acid (SA) is an industrially important chiral compound used in diverse commercial applications, and the insufficient supply by isolation from plants and expensive chemical synthesis of SA has increased the importance of developing strategies for SA synthesis. In our previous studies, glycerol was observed to be an effective carbon source for SA accumulation in E. coli DHPYAAS-T7, where the PTS operon (ptsHIcrr) and aroL and aroK genes were inactivated, and the tktA, glk, aroE, aroF fbr , and aroB genes were overexpressed. For further investigation of the effects of glycerol aerobic fermentation on SA accumulation in E. coli BL21(DE3), the glpD, glpK genes and tktA, glk, aroE, aroF fbr , aroB genes were overexpressed simultaneously. The results indicated that SA production was increased 5.6-fold, while the yield was increased 5.3-fold over that of parental strain in shake flasks. It is demonstrated that the aerobic fermentation of glycerol associated with glpD and glpK gene overexpression increased glycerol flux, resulting in higher SA accumulation in E. coli BL21(DE3)-P-DK.  相似文献   

6.
Summary A cytochrome containing fraction virtually devoid of the photosynthetic apparatus (bacteriochlorophyll and/or chromatophores) was isolated from Rps. palustris grown photolithotrophically with S2O3 =as the exogenous electron donor. This fraction contained predominantly cytochromes of c, a and o type and exhibited thiosulfate: cytochrome c oxidoreductase and ferrocytochrome c:O2 oxidoreductase activities. Under anaerobic conditions the enzyme preparation catalyzed an ATP-dependent NAD+ reduction by S2O3 =in the dark involving a reversal of electron transfer from cytochrome c and yielding a molar stoichiometry of approximately 2:1 for the ferrocytochrome c oxidized and NAD+ reduced. In this process approximately 4 to 7 molar equivalents of ATP were utilized/equivalent of NAD+ reduced. The optimal reaction occurred at pH 8.0 and in the presence of 55 M added mammalian cyt. c, 1.7 mM Mg++, 1.7 mM ATP and 7.0 mM S2O3 =. The S2O3 =-linked ATP-driven reduction of NAD+ as well as the coupled oxidation of cyt. c were inhibited completely by 5 m CCCP or 10 M DNP and the reaction was also markedly sensitive to other uncouplers of the energy transfer reactions. The pathway of electron transfer from S2O3 = to NAD+ appears to involve cyt. c, b, and flavoprotein systems as evidenced by the complete inhibition of the process by low concentrations of antimycin A, NOQNO, rotenone and amytal.Non-standard abbreviations BAL British Anti-Lewisite (2,3-Dimercaptopropanol) - CCCP Carbonyl-cyanide-m-chlorophenylhydrazone - DBP 2,6-Dibromophenol - DNP 2,4-Dinitrophenol - EDTA Ethylenediamine tetraacetic acid - GSH reduced glutathione - NOQNO 2-n-Nonyl-4-hydroxyquinoline-N-oxide - PCP Pentachlorophenol - PABA p-aminobenzoic acid. Post doctorate fellow of the Deutsche Forschungsgemeinschaft  相似文献   

7.
Seedlings ofPicea sitchensis, Thuja plicata andTsuga heterophylla were supplied N hydroponically at one of four exponentially increasing rates of addition (0.09, 0.07, 0.05, or 0.025 gN-1 day-1) for up to 3 months in a naturally illuminated glasshouse. Relative growth rates (RGR) were analyzed as a function of N uptake, the allocation of assimilated N to foliage (LNFR), foliar N concentrations (Nla) and met assimilation rates (NAR), which were combined to estimate N productivity (RGR per unit whole-plant N concentration). Nitrogen accumulation, biomass and N partitioning and RGR and its components varied with species in response to the different N regimes.T. heterophylla had the lowest maximum wholeplant N concentrations (wpN) and specific absorption rates for N and exhibited the least plasticity in root: shoot ratios as wpN increased from 11–21 mg g-1. In all species, RGR increased linearly with wpN, while LNFR increased curvilinearly. Foliar N (Nla) increased linearly with wpN and NAR increased linearly with Nla. The RGRs ofT. heterophylla were highest at wpNs up to 18 mg g-1, a result of higher foliar N use efficiencies (NAR/Nla). However, RGR increased more with wpN inT. plicata andP. sitchensis. Although LNFR increased with wpN in all species, foliar N use efficiency declined, possibly due to an increased partitioning of foliar soluble N to non-photosynthetic compounds. Thus, in each species, N productivity did not increase above intermediate levels of wpN: 14 mg g-1 inT. heterophylla, 16 mg g-1 inP. sitchensis and 17 mg g-1 inT. plicata.  相似文献   

8.
9.
Nutritional Requirements of Methanosarcina sp. Strain TM-1   总被引:2,自引:1,他引:1       下载免费PDF全文
Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO2, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl2 · 2H2O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO2 for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO2. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH4+ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH4+ limitation.  相似文献   

10.
Glutathione reductase (GR; E.C. 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). In this study we tested the effects of Al3+, Ba2+, Ca2+, Li+, Mn2+, Mo6+, Cd2+, Ni2+, and Zn2+ on purified bovine liver GR. In a range of 10?μM–10?mM concentrations, Al3+, Ba2+, Li+, Mn2+, and Mo6+, and Ca2+ at 5?μM–1.25?mM, had no effect on bovine liver GR. Cadmium (Cd2+), nickel (Ni2+), and zinc (Zn2+) showed inhibitory effects on this enzyme. The obtained IC50 values of Cd2+, Ni2+, and Zn2+ were 0.08, 0.8, and 1?mM, respectively. Cd2+ inhibition was non-competitive with respect to both GSSG (KiGSSG 0.221?±?0.02?mM) and NADPH (KiNADPH 0.113?±?0.008?mM). Ni2+ inhibition was non-competitive with respect to GSSG (KiGSSG 0.313?±?0.01?mM) and uncompetitive with respect to NADPH (KiNADPH 0.932?±?0.03?mM). The effect of Zn2+ on GR activity was consistent with a non-competitive inhibition pattern when the varied substrates were GSSG (KiGSSG 0.320?±?0.018?mM) and NADPH (KiNADPH 0.761?±?0.04?mM), respectively.  相似文献   

11.
For the high production of phenylalanine by Escherichia coli, we cloned the pheAFR and aroFFR genes (FR = feedback resistant), which encoded chorismate mutase P-prephenate dehydratase and 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase that are feedback inhibition-free as to the endproducts, into a temperature-controllable expression vector composed of the PR and PL promoter and a temperature sensitive repressor, cI857, of bacteriophage lambda. The plasmid obtained was designated as pSY130-14, and the temperature dependency of expression of the cloned genes and of phenylalanine production was investigated at different temperatures between 30 and 42°C using the strain AT2471 harbouring the plasmid. Above 35°C, the pheAFR gene and aroFFR gene expressions, and activities of both enzymes continued to increase up to 42°C. The cell concentration remained constant up to 38.5°C, but started to decrease sharply above 40°C, while the cell concentration of the host strain, AT2471, remained constant at all temperatures tested. The concentration of phenylalanine also depended on the temperature, and the highest production of phenylalanine, 18.6 g l−1, was obtained from glucose at 38.5°C in a 2.5 1 reactor.  相似文献   

12.
2-Deoxy-d -glucose (2 DG) entered synaptosomes (from rat brain) by a high-affinity, Na+-independent glucose transport system with a Km, of 0.24 mM. 3-O-methyl-glucose, D-glucose, and phloretin were competitive inhibitors of 2-DG transport with Ki's of 7 mM, 64 μM, and 0·75 μM, respectively. Insulin was without effect. 2-DG uptake was also saturable at high substrate concentrations with an apparent low affinity Km, of 75 mM, where the Kl, for glucose was 17.5 mM. We are not certain whether the rate-limiting step for the low-affinity uptake system is attributable to transport or phosphorylation. However, the high-affinity glucose transport system probably is a special property of neuronal cell membranes and could be useful in helping to distinguish separated neurons from glial cells.  相似文献   

13.
(R)-1,3-butanediol ((R)-1,3-BD) is an important substrate for the synthesis of industrial chemicals. Despite its large demand, a bioprocess for the efficient production of 1,3-BD from renewable resources has not been developed. We previously reported the construction of recombinant Escherichia coli that could efficiently produce (R)-1,3-BD from glucose. In this study, the fermentation conditions were optimized to further improve 1,3-BD production by the recombinant strain. A batch fermentation was performed with an optimized overall oxygen transfer coefficient (82.3?h?1) and pH (5.5); the 1,3-BD concentration reached 98.5?mM after 36?h with high-yield (0.444?mol (mol glucose)?1) and a high maximum production rate (3.63?mM?h?1). In addition, a fed-batch fermentation enabled the recombinant strain to produce 174.8?mM 1,3-BD after 96?h cultivation with a yield of 0.372?mol (mol glucose)?1, a maximum production rate of 3.90?mM?h?1, and a 98.6% enantiomeric excess (% ee) of (R)-1,3-BD.  相似文献   

14.
Glucose is a primary stimulator of insulin secretion in pancreatic β-cells. High concentration of glucose has been thought to exert its action solely through its metabolism. In this regard, we have recently reported that glucose also activates a cell-surface glucose-sensing receptor and facilitates its own metabolism. In the present study, we investigated whether glucose activates the glucose-sensing receptor and elicits receptor-mediated rapid actions. In MIN6 cells and isolated mouse β-cells, glucose induced triphasic changes in cytoplasmic Ca2+ concentration ([Ca2+]c); glucose evoked an immediate elevation of [Ca2+]c, which was followed by a decrease in [Ca2+]c, and after a certain lag period it induced large oscillatory elevations of [Ca2+]c. Initial rapid peak and subsequent reduction of [Ca2+]c were independent of glucose metabolism and reproduced by a nonmetabolizable glucose analogue. These signals were also blocked by an inhibitor of T1R3, a subunit of the glucose-sensing receptor, and by deletion of the T1R3 gene. Besides Ca2+, glucose also induced an immediate and sustained elevation of intracellular cAMP ([cAMP]c). The elevation of [cAMP]c was blocked by transduction of the dominant-negative Gs, and deletion of the T1R3 gene. These results indicate that glucose induces rapid changes in [Ca2+]c and [cAMP]c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor.  相似文献   

15.
The purpose of this study was to investigate criteria for maximal effort in middle-aged men and women undertaking a maximal exercise test until they were exhausted if no measurements of oxygen uptake are made. A large group of 2164 men and 975 women, all active in sports and aged between 40 and 65 years, volunteered for a medical examination including a progressive exercise test to exhaustion on a cycle ergometer. In the 3rd min of recovery a venous blood sample was taken to determine the plasma lactate concentration ([la]p, 3min). Lactate concentration and maximal heart rate (f c, max) were lower in the women than in the men (P<0.001). Multiple regression analyses were performed to assess the contribution of sex to [la]p, 3 min, independent of age and f c max, It was found that [la]p,3 min was about 2.5 mmol·l–1 lower in women than in men of the same age and f c, max. In our population 88% of the men and 85% of the women met a combination of the following f c, max and [la]p, 3min criteria: f c, max equal to or greater than 220 minus age beats·min–1 and/or [la]p, 3min equal to or greater than 8 mmol·l–1 in the men and f c, max equal to or greater than 220 minus age beats·min–1 and/or [la]p, 3min equal to or greater than 5.5 mmol·1–1 in the women.  相似文献   

16.
Summary Cell-free preparations from T. neapolitanus catalyzed an ATP-dependent reduction of pyridine nucleotides by thiosulfate. The reduction of flavins by thiosulfate was also observed to be an energy-linked process. Optimal reaction occurred at pH 7.3–7.5 in the presence of 7 mM S2O3 =, 1.5 mM ATP and 0.7 mM NAD+ or NADP+. The enzyme(s) catalyzing the energy-linked reactions appear to reside in the 144000 x g supernatant fraction since washed particles failed to catalyze the ATP driven NAD+ reduction by S2O3 +; the cell-free preparations contained, however, S2O3 = oxidase and ferro-cytochrome c: O2 oxidoreductase activities. The ATP-driven reduction of flavins or that of the pyridine nucleotides was inhibited bythe inhibitors that intersect the electron transport chain in the flavin or that of the cytochrome b and c regions. In the flavin-inhibited system, quinones could substitute as electron bypass carriers for the reduction of pyridine nucleotides. Uncouplers of oxidative phosphorylation and oligomycin inhibited the energy-transfer reactions. A utilization of 2 to 3 ATP equivalents was observed for the reduction of each equivalent of NAD+. Such observations indicate that the T. neapolitanus system operated with an efficiency of approximately 80% with respect to the utilization of energy for the generation of reducing power.Non-standard abbreviations HQNO 2-n-hyptyl-4-hydroxyquinoline N-oxide - TTFA Thenoyl triflouroacetone - CCCP m-chlorocarbonylcyanide-phenylhydrazone - DNP 2,4-dinitrophenol  相似文献   

17.
Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.  相似文献   

18.
Various inhibitors were tested for their inhibitory effects on soybean urease. The Ki values for boric acid, 4-bromophenylboronic acid, butylboronic acid, and phenylboronic acid were 0.20?±?0.05?mM, 0.22?±?0.04?mM, 1.50?±?0.10?mM, and 2.00?±?0.11?mM, respectively. The inhibition was competitive type with boric acid and boronic acids. Heavy metal ions including Ag+, Hg2+, and Cu2+ showed strong inhibition on soybean urease, with the silver ion being a potent inhibitor (IC50 = 2.3?×?10?8 mM). Time-dependent inhibition studies exhibited biphasic kinetics with all heavy metal ions. Furthermore, inhibition studies with sodium salts of mineral acids (NaF, NaCl, NaNO3, and Na2SO4) showed that only F? inhibited soybean urease significantly (IC50 = 2.9?mM). Competitive type of inhibition was observed for this anion with a Ki value of 1.30?mM.  相似文献   

19.

Background  

Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroG fbr, tktA, aroB and aroE, on SA synthesis.  相似文献   

20.
A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (E m7 = –254 mV) and one low-spin, high-potential (E m7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c 555 as the electron donor, the K M and V max for peroxide reduction were 510 ± 100 nM and 425 ± 22 mol ferrocytochrome c 555 oxidized min–1 (mole cytochrome c peroxidase)–1, respectively. Received: 6 January 1997 / Accepted: 27 May 1997  相似文献   

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