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1.
Neves AR Ventura R Mansour N Shearman C Gasson MJ Maycock C Ramos A Santos H 《The Journal of biological chemistry》2002,277(31):28088-28098
The involvement of nicotinamide adenine nucleotides (NAD(+), NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using (13)C and (31)P NMR to monitor in vivo the kinetics of the pools of NAD(+), NADH, ATP, inorganic phosphate (P(i)), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of (13)C-labeled NAD(+) and NADH. The capacity of L. lactis MG1363 to regenerate NAD(+) was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH(-) deficient strain was constructed by double crossover. Upon supply of glucose, NAD(+) was constant and maximal (approximately 5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH(-) strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH(-) strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD(+). However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P(i) content could play an important role. 相似文献
2.
Isolation and properties of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 mutants producing diacetyl and acetoin from glucose. 总被引:2,自引:0,他引:2
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Following treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine, three mutants of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 that produced diacetyl and acetoin from glucose were isolated. The lactate dehydrogenase activity of these mutants was strongly attenuated, and the mutants produced less lactate than the parental strain. The kinetic properties of lactate dehydrogenase of strain CNRZ 483 and the mutants revealed differences in the affinity of the enzyme for pyruvate, NADH, and fructose-1,6-diphosphate. When cultured aerobically, strain CNRZ 483 transformed 2.3% of glucose to acetoin and produced no diacetyl or 2,3-butanediol. Under the same conditions, mutants 483L1, 483L2, and 483L3 transformed 42.0, 78.9, and 75.8%, respectively, of glucose to C4 compounds (diacetyl, acetoin, and 2,3-butanediol). Anaerobically, strain CNRZ 483 produced no C4 compounds, while mutants 483L1, 483L2, and 483L3 transformed 2.0, 37.0, and 25.8% of glucose to acetoin and 2,3-butanediol. In contrast to the parental strain, the NADH balance showed that the mutants regenerated most of the NAD via NADH oxidase under aerobic conditions and by ethanol production under anaerobic conditions. 相似文献
3.
4.
IS981-mediated adaptive evolution recovers lactate production by ldhB transcription activation in a lactate dehydrogenase-deficient strain of Lactococcus lactis 总被引:1,自引:0,他引:1
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Bongers RS Hoefnagel MH Starrenburg MJ Siemerink MA Arends JG Hugenholtz J Kleerebezem M 《Journal of bacteriology》2003,185(15):4499-4507
5.
A R Neves A Ramos C Shearman M J Gasson J S Almeida H Santos 《European journal of biochemistry》2000,267(12):3859-3868
The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed. 相似文献
6.
Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate reductase (GRHPR) has a potentially protective role metabolising glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for glyoxylate and related substrates. The Km values and specificity constants (Kcat/K(M)) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of glyoxylate and hydroxypyruvate. K(M) values with glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of glyoxylate, K(M) values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD+ as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH (K(M) 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed. 相似文献
7.
Skory CD 《Applied and environmental microbiology》2000,66(6):2343-2348
8.
Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production 总被引:3,自引:0,他引:3
The effects of initial glucose concentration and calcium lactate concentration on the lactic acid production by the parent strain, Lactobacillus lactis BME5-18, were studied. The results of the experiments indicated that glucose and lactate repressed the cell growth and the lactic acid production by Lactobacillus lactis BME5-18. A L(+)-lactic acid overproducing strain, Lactobacillus lactis BME5-18M, was screened by mutagenizing the parent strain with ultraviolet (UV) light irradiation and selecting the high glucose and lactate calcium concentration repression resistant mutant. Starting with a concentration of 100g L(-1) glucose, the mutant produced 98.6 g L(-1) lactic acid after 60 h in flasks, 73.9% higher than that of the parent strain. The L(+)-lactic acid purity was 98.1% by weight based on the amount of total lactic acid. The culture of the parent strain could not be analyzed well by conventional metabolic flux analysis techniques, since some pyruvate were accumulated intracellularly. Therefore, a revised flux analysis method was proposed by introducing intracellular pyruvate pool. Further studies demonstrate that there is a high level of NADH oxidase activity (12.11 mmol mg(-1) min(-1)) in the parent strain. The molecular mechanisms of the strain improvement were proposed, i.e., the high level of NADH oxidase activity was eliminated and the uptake rate of glucose was increased from 82.1 C-mmol (g DW h)(-1) to 98.9 C-mmol (g DW h)(-1) by mutagenizing the parent strain with UV, and therefore the mutant strain converts mostly pyruvate to lactic acid with a higher productivity (1.76 g L(-1) h(-1)) than the parent strain (0.95 g L(-1) h(-1)). 相似文献
9.
Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H(2)O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H(2)O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15 ± 0.08 mM to 9.94 ± 0.07 mM, and the corresponding diacetyl production increased from 1.07 ± 0.03 mM to 4.16 ± 0.06 mM with the intracellular NADH/NAD(+) ratios varying from 0.711 ± 0.005 to 0.383 ± 0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD(+) ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H(2)O-forming NADH oxidase activity led to 76.95% lower H(2)O(2) concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H(2)O(2) accumulation and prolong cell survival. 相似文献
10.
Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae 总被引:3,自引:0,他引:3
A mitochondrial NADH:Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B. By this procedure a 2000-fold purification is achieved with respect to whole cells or a 150-fold purification with respect to the mitochondrion. The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/polyacrylamide gel electrophoresis. The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.6 = -370 mV and no iron-sulphur clusters. The enzyme is specific for NADH with apparent Km = 31 microM and was found to be inhibited by flavone (I50 = 95 microM), but not by rotenone or piericidin. The purified enzyme can use ubiquinone-2, -6 or -10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates. The greatest turnover of NADH was obtained with ubiquinone-2 as acceptor (2500 s-1). With the natural ubiquinone-6 this value was 500 s-1. The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5-9.0. The amount of enzyme in the cell is subject to glucose repression; it increases slightly when cells, grown on glucose or lactate, enter the stationary phase. The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH. The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed. 相似文献
11.
V I Loushchak 《Biochemistry international》1992,26(5):905-912
Purified cytoplasmic and membrane-bound lactate dehydrogenases (LDH) from white muscle of skate were characterized, Km for pyruvate and NADH for purified LDH were 150 +/- 16 and 29 +/- 7 microM, and for membrane-bound LDH were 185 +/- 22 and 7.5 +/- 1.5 microM, respectively. The membrane-bound enzyme was not inhibited by high pyruvate concentration (up to 20 mM) in contrast to purified LDH. Part of membrane-bound LDH was released by incubation in solutions with a high level of KCl (up to 1 M) or at alkaline pH. The inactivation rate during trypsin digestion for solubilized LDH was 2-3-fold higher than that for the membrane-bound enzyme. 相似文献
12.
Immunochemical evidence that the single lactate dehydrogenase of lampreys is more similar to LDHB4 than to LDHA4 of hagfish 总被引:2,自引:0,他引:2
The tetrameric lactate dehydrogenases (LDH) of vertebrates contain several different subunits that arose by gene duplication. While the A and B subunits occur in all classes of gnathostomes, the enzymes of agnathans appear to represent two stages in the evolution of vertebrate LDH. Lampreys of the family Petromyzontidae have a single enzyme classified as LDHA4, while hagfish possess both A and B subunits which form only the two homopolymers LDHA4 and LDHB4. It is generally assumed that the original vertebrate LDH was an A4 type, that duplication to give the B subunit occurred prior to the divergence of lampreys and hagfish, and that modern lampreys subsequently lost expression of the B gene. Lactate dehydrogenases were purified from representatives of all three lamprey families, and it was confirmed that members of the Mordaciidae and Geotriidae also possess single tetrameric LDH enzymes containing one subunit type. The kinetic properties of the lamprey LDH enzymes were compared with the LDH homopolymers of hagfish, skate, and sardine. These properties did not allow the lamprey enzymes to be unequivocally identified as either LDHA4 or LDHB4. Immunochemical titration using antisera against lamprey and hagfish LDH homopolymers demonstrated that the lamprey LDH enzymes showed greater immunochemical similarity to LDHB4 than to LDHA4 of hagfish. It is concluded that there is little evidence for the claim that the original vertebrate LDH was an A4 rather than B4 type. 相似文献
13.
M M Bianchi L Brambilla F Protani C L Liu J Lievense D Porro 《Applied and environmental microbiology》2001,67(12):5621-5625
A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using a Kluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene (KlPDC1) and transformed with the heterologous lactate dehydrogenase gene (LDH). The LDH gene was placed under the control the KlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g(-1), suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used K. lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1alpha subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g(-1) (maximum theoretical yield, 1 g g(-1)), and with high LDH activity. 相似文献
14.
Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation. 总被引:13,自引:0,他引:13
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The L-(+)-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) of Streptococcus lactis C10, like that of other streptococci, was activated by fructose 1,6-diphosphate (FDP). The enzyme showed some activity in the absence of FDP, with a pH optimum of 8.2; FDP decreased the Km for both pyruvate and reduced nicotinamide adenine dinucleotide (NADH) and shifted the pH optimum to 6.9. Enzyme activity showed a hyperbolic response to both NADH and pyruvate in all the buffers tried except phosphate buffer, in which the response to increasing NADH was sigmoidal. The FDP concentration required for half-maximal velocity (FDP0.5V) was markedly influenced by the nature of the assay buffer used. Thus the FDP0.5V was 0.002 mM in 90 mM triethanolamine buffer, 0.2 mM in 90 mM tris(hydroxymethyl)aminomethanemaleate buffer, and 4.4 mM in 90 mM phosphate buffer. Phosphate inhibition of FDP binding is not a general property of streptococcal lactate dehydrogenase, since the FDP0.5V value for S. faecalis 8043 lactate dehydrogenase was not increased by phosphate. The S. faecalis and S. lactis lactate dehydrogenases also differed in that Mn2+ enhanced FDP binding in S. faecalis but had no effect on the S. lactis dehydrogenase. The FDP concentration (12 to 15 mM) found in S. lactis cells during logarithmic growth on a high-carbohydrate (3% lactose) medium would be adequate to give almost complete activation of the lactate dehydrogenase even if the high FDP0.5V value found in 90 mM phosphate were similar to the FDP requirement in vivo. 相似文献
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16.
Neves AR Ramos A Costa H van Swam II Hugenholtz J Kleerebezem M de Vos W Santos H 《Applied and environmental microbiology》2002,68(12):6332-6342
17.
Wisselink HW Mars AE van der Meer P Eggink G Hugenholtz J 《Applied and environmental microbiology》2004,70(7):4286-4292
To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production. 相似文献
18.
Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase 总被引:9,自引:0,他引:9
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Felix Lopez de Felipe Michiel Kleerebezem Willem M. de Vos Jeroen Hugenholtz 《Journal of bacteriology》1998,180(15):3804-3808
NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2 gene, which encodes the H2O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L. lactis nisA promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via α-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD+ ratios in the cells. 相似文献
19.
The chain oxidation of lactate dehydrogenase-bound NADH initiated by superoxide radicals and propagated by oxygen was studied with pulse radiolysis. The kinetic parameters were re-evaluated in a system with carefully purified reagents (water and other chemicals) and in the presence of EDTA. The rate constant for the oxidation of the enzyme-bound NADH by O2- is calculated from the observed pseudo-first order disappearance of NADH and the chain length (molecules of NADH oxidized per O2- anion generated in the pulse). It is (1.0 +/- 0.2) X 10(5) M-1 S-1, consistent within a 13-fold variation in lactate dehydrogenase. NADH complex concentration and with varying chain length up to 6.1. Based on experiments with varying pH values from 4.5 to 9.0, the rate constant for oxidation of enzyme-bound NADH by HO2 is estimated to be 2.0 X 10(6) M-1 S-1. 相似文献
20.
Caldicellulosiruptor saccharolyticus displays superior H2 yields on a wide range of carbon sources provided that lactate formation is avoided. Nevertheless, a low lactate flux is initiated as the growth rate declined in the transition to the stationary phase, which coincides with a drastic decrease in the glucose consumption and acetate production fluxes. In addition, the decrease in growth rate was accompanied by a sudden increase and then decrease in NADH levels. The V′MAX of the lactate dehydrogenase (LDH) doubled when the cells entered the stationary phase. Kinetic analysis revealed that at the metabolic level LDH activity is regulated through (i) competitive inhibition by pyrophosphate (PPi, ki=1.7 mM) and NAD (ki=0.43 mM) and (ii) allosteric activation by FBP (300%), ATP (160%) and ADP (140%). From these data a MWC-based model was derived. Simulations with this model could explain the observed lactate shift by displaying how the sensitivity of LDH activity to NADH/NAD ratio varied with different PPi concentrations. Moreover, the activation of LDH by ATP indicates that C. saccharolyticus uses LDH as a means to adjusts its flux of ATP and NADH production. To our knowledge, this is the first time PPi is observed as an effector of LDH. 相似文献