Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum

A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.     

Metabolic Consequences of Altered PhosphoenolpyruvateCarboxykinase Activity in Corynebacterium glutamicum Reveal Anaplerotic Regulation Mechanisms in Vivo

Corynebacterium glutamicum possesses high in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase (PEPCk) during growth on glucose, resulting together with anaplerotic carboxylation reactions in a PEP/pyruvate/oxaloacetate substrate cycle. The present study investigated the changes in intracellular fluxes and metabolite concentrations that are caused by altered PEPCk activity in L-lysine-producing C. glutamicum MH20-22B, applying a recently developed (13)C labeling-based strategy for anaplerotic flux resolution and quantification. Abolition of PEPCk activity by deletion of the respective pck gene resulted in increased intracellular concentrations of oxaloacetate L-aspartate, alpha-ketoglutarate, pyruvate, and L-lysine and in a 60% enhanced flux toward L-lysine biosynthesis, whereas increasing the PEPCk activity by pck overexpression had opposite effects. The results of the combined measurements of enzyme activities, in vivo fluxes, and metabolite concentrations were exploited to elucidate the in vivo regulation of anaplerotic reactions in C. glutamicum, and implications for the metabolic engineering of amino-acid-producing strains are discussed.     

Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-defective mutants.

We found that the simple addition of L-methionine to the wild type of Corynebacterium glutamicum results in excretion of the cellular building block L-lysine up to rates of 2.5 nmol/min/mg (dry weight). Biochemical analyses revealed that L-methionine represses the homoserine dehydrogenase activity and reduces the intracellular L-threonine level from 7 to less than 2 mM. Since L-lysine synthesis is regulated mainly by L-threonine (plus L-lysine) availability, the result is enhanced flux towards L-lysine. This indicates a delicate and not well controlled type of flux control at the branch point of aspartate semialdehyde conversion to either L-lysine or L-threonine, probably due to the absence of isoenzymes in C. glutamicum. The inducible system of L-lysine excretion discovered was used to isolate mutants defective in the excretion of this amino acid. One such mutant characterized in detail accumulated 174 mM L-lysine in its cytosol without extracellular excretion of L-lysine, whereas the wild type accumulated 53 mM L-lysine in the cytosol and 5.9 mM L-lysine in the medium. The mutant was unaffected in L-lysine uptake or L-isoleucine or L-glutamate excretion, and also the membrane potential was unaltered. This mutant therefore represents a strain with a defect in an excretion system for the primary metabolite L-lysine.     

Metabolic characterization of a L-lysine-producing strain by continuous culture

Continuous culture experiments with the L-producer, Corynebacterium glutamicum, were carried out to characterize the effect of specific growth rate on fermentation yields, specific rates, productivities, and fluxes through the primary metabolism. The specific productivity of L-lysine exhibited a maximum with respect to specific growth rate, with an initial growth-associated behavior up to specific growth rates of about 0.1 h(-1), and a constant specific productivity for specific growth rates in the range of about 0.1 to 0.2 h(-1). The productivity dropped at specific growth rates larger than about 0.2 h(-1). The yield of L-lysine on glucose increased approximately linearly with decreasing specific growth rate over the entire range studied, as did the respiratory quotient. A direct relationship was established between the culture respiratory quotient and the L-lysine yield. By explicitly accounting for glucose used for biomass synthesis, it was shown that the strain synthesizes L-lysine with an intrinsic yield, or efficiency, of about 0.41 mol L-lysine/mol glucose, compared with the theoretical yield of 0.75 mol/mol. Metabolic flux modeling based on the continuous culture data suggests that the production of ATP is not likely to be a limiting factor in L-lysine production, and that a high TCA cycle activity, coupled with a tightly controlled split of metabolite flow at the PEP node, is likely the cause of the large discrepancy between theoretical and actual yields in L-lysine fermentations.     

Role of the Bacillus methanolicus citrate synthase II gene,citY, in regulating the secretion of glutamate in L-lysine-secreting mutants

The thermotolerant, restrictive methylotroph Bacillus methanolicus MGA3 (ATCC 53907) can secrete 55 g of glutamate per liter (maximum yield, 0.36 g/g) at 50 degrees C with methanol as a carbon source and a source of ammonia in fed-batch bioreactors. A homoserine dehydrogenase mutant, 13A52-8A66, secreting up to 35 g of L-lysine per liter in fed-batch fermentations had minimal 2-oxoglutarate dehydrogenase activity [7.3 nmol min(-1) (mg of protein)(-1)], threefold-increased pyruvate carboxylase activity [535 nmol min(-1) (mg of protein)(-1)], and elevated citrate synthase (CS) activity [292 nmol min(-1) (mg of protein)(-1)] and simultaneously secreted glutamate (20 to 30 g per liter) and L-lysine. The flow of carbon from oxaloacetate is split between transamination to aspartate and formation of citrate. To investigate the regulation of this branch point, the B. methanolicus gene citY encoding a CSII protein with activity at 50 degrees C was cloned from 13A52-8A66 into a CS-deficient Escherichia coli K2-1-4 strain. A citY-deficient B. methanolicus mutant, NCS-L-7, was also isolated from the parent strain of 13A52-8A66 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, followed by selection with monofluoroacetate disks on glutamate plates. Characterization of these strains confirmed that citY in strain 13A52-8A66 was not altered and that B. methanolicus possessed several forms of CS. Analysis of citY cloned from NCS-L-7 showed that the reduced CS activity resulted from a frameshift mutation. The level of glutamate secreted by NCS-L-7 was reduced sevenfold and the ratio of L-lysine to glutamate secreted was increased 4.5-fold compared to the wild type in fed-batch cultures with glutamate feeding. This indicates that glutamate secretion in L-lysine-overproducing mutants can be altered in favor of increased L-lysine secretion by regulating in vivo CS activity.     

The effect of zearalenone on PSII photochemical activity and growth in wheat and soybean under salt (NaCl) stress

The effects of mycotoxin zearalenone (ZEN) on the photochemical activity of photosystem II (PSII) in wheat and soybean leaf discs incubated in ZEN solutions as well as the after-effects of pre-sowing soaking of seeds in solutions containing ZEN on the photochemical activity of PSII and on the seedlings growth under salt stress (NaCl solutions were investigated). The incubation of wheat leaf discs in ZEN solutions strongly inhibited the energy flux per cross section (CS) for absorption (ABS/CS), trapping (TRo/CS) and electron transport (ETo/CS), while the effects of ZEN action on soybean discs were opposite and the values of those parameters significantly increased with the increase in ZEN concentration. Incubation of seeds in a ZEN solution resulted in an increase in photochemical efficiency of PSII in soybean seedlings, but did not induce any response of PSII in those of wheat at medium illuminations. Only at the stronger illumination for both species did ZEN induce an increase in efficiency of excitation energy capture by open PSII reaction centers, photochemical quenching of chlorophyll a fluorescence and quantum yield of PSII electron transport. Pre-sowing soaking of seeds in a ZEN solution decreased the photoinhibitory injuries of PSII in wheat and soybean due to safe scattering of the excess excitation energy through an increase in energy-dependent quenching (qE) and state transition quenching (qT). ZEN when added to NaCl solutions during the period of germination contributed to reduction in the growth inhibition of wheat seedlings. The incubation of wheat leaf discs in ZEN solutions strongly inhibited CS, ABS/CS, TRo/CS and ETo/CS. Possible effects of ZEN on some physiological processes in plants have been discussed especially in the context with photochemical activity of PSII and a salt stress.     

Reaction engineering analysis of L-lysine transport by Corynebacterium glutamicum

To identify potential L-lysine export limitations by Corynebacterium glutamicum in the L-lysine production process, the excretion of L-lysine was studied in continuous and fed-batch operated stirred tank reactors. A structured biochemical model of the L-lysine excretion mechanism was used to determine the activity of the export carrier and to calculate a cell-specific concentration of the export carrier. For the biochemical characterization of this specific carrier concentration a standardized L-lysine efflux test was developed. Carrier activity, cell-specific carrier concentration, and the specific L-lysine export rate were identified as a function of pH value and L-lysine concentration in the reactors. Also, the correlation of these parameters to the metabolic state of C. glutamicum was determined. The pH value in the reactor governs the carrier activity (maximum at pH 6.5) and the specific carrier concentration (maximum at pH 8.0). The specific L-lysine export rate, as the product of carrier activity and specific carrier concentration, revealed a maximum at pH 7.0. Decreasing L-lysine productivities also correlated with decreasing specific carrier concentrations. The L-lysine concentration in the reactor had no effect on the specific carrier concentration but strongly inhibited the carrier activity. The specific export rate was reduced to 50% at 400 mM L-lysine compared to the specific export rate at 80 mM L-lysine. (c) 1996 John Wiley & Sons, Inc.     

Pathway analysis and metabolic engineering in Corynebacterium glutamicum

The gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of L-glutamate and L-lysine. During the last 15 years, genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for the construction of strains with the desired genotypes. In order to obtain a better understanding of the central metabolism and to quantify the in vivo fluxes in C. glutamicum, the [13C]-labelling technique was combined with metabolite balancing to achieve a unifying comprehensive pathway analysis. These methods can determine the flux distribution at the branch point between glycolysis and the pentose phosphate pathway. The in vivo fluxes in the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified glucose-6-phosphate and 6-phosphogluconate dehydrogenases determined in vitro were in full accordance with the fluxes measured by the [13C]-labelling technique. These data indicate that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH/NADP concentrations and the specific activity of glucose-6-phosphate dehydrogenase. The carbon flux via the oxidative pentose phosphate pathway correlated with the NADPH demand for L-lysine synthesis. Although it has generally been accepted that phosphoenolpyruvate carboxylase fulfills a main anaplerotic function in C. glutamicum, we recently detected that a biotin-dependent pyruvate carboxylase exists as a further anaplerotic enzyme in this bacterium. In addition to the activities of these two carboxylases three enzymes catalysing the decarboxylation of the C4 metabolites oxaloacetate or malate are also present in this bacterium. The individual flux rates at this complex anaplerotic node were investigated by using [13C]-labelled substrates. The results indicate that both carboxylation and decarboxylation occur simultaneously in C. glutamicum so that a high cyclic flux of oxaloacetate via phosphoenolpyruvate to pyruvate was found. Furthermore, we detected that in C. glutamicum two biosynthetic pathways exist for the synthesis of DL-diaminopimelate and L-lysine. As shown by NMR spectroscopy the relative use of both pathways in vivo is dependent on the ammonium concentration in the culture medium. Mutants defective in one pathway are still able to synthesise enough L-lysine for growth, but the L-lysine yields with overproducers were reduced. The luxury of having these two pathways gives C. glutamicum an increased flexibility in response to changing environmental conditions and is also related to the essential need for DL-diaminopimelate as a building block for the synthesis of the murein sacculus.     

From Metabolomics to Fluxomics: A Computational Procedure to Translate Metabolite Profiles into Metabolic Fluxes

We describe a believed-novel procedure for translating metabolite profiles (metabolome) into the set of metabolic fluxes (fluxome) from which they originated. Methodologically, computational modeling is integrated with an analytical platform comprising linear optimization, continuation and dynamic analyses, and metabolic control. The procedure was tested with metabolite profiles obtained from ex vivo mice Langendorff-heart preparations perfused with glucose. The metabolic profiles were analyzed using a detailed kinetic model of the glucose catabolic pathways including glycolysis, pentose phosphate (PP), glycogenolysis, and polyols to translate the glucose metabolome of the heart into the fluxome. After optimization, the ability of the model to simulate the initial metabolite profile was confirmed, and metabolic fluxes as well as the structure of control and regulation of the glucose catabolic network could be calculated. We show that the step catalyzed by phosphofructokinase together with ATP demand and glycogenolysis exert the highest control on the glycolytic flux. The negative flux control exerted by phosphofructokinase on the PP and polyol pathways revealed that the extent of glycolytic flux directly affects flux redirection through these pathways, i.e., the higher the glycolytic flux the lower the PP and polyols. This believed-novel methodological approach represents a step forward that may help in designing therapeutic strategies targeted to diagnose, prevent, and treat metabolic diseases.     

Hepatic metabolite profiles in mice with a suboptimal selenium status

Selenium is an essential trace element and mediates its functions via various selenoproteins such as glutathione peroxidases or thioredoxin reductases. A suboptimal selenium supply causes metabolic disturbances and is associated with an increased risk to develop different disorders, including cancer or cardiovascular diseases. This study aimed to assess the impact of a suboptimal selenium status on the hepatic metabolome of male mice analyzed by a targeted liquid chromatography/tandem mass spectrometry and a method based on non-targeted gas chromatography hyphenated with mass spectrometry. Feeding animals a diet with about half of the recommended selenium content supplied as selenomethionine caused liver glutathione peroxidase and thioredoxin reductase activities to decline and lipid peroxidation to increase. Serum T₃ thyroid hormone concentration also declined via a reduced hepatic deiodinase activity. Metabolite profiling revealed predominantly changes in cysteine and carbon-1 metabolism as well as in selected lipid subclasses. In particular the concentrations of palmitoylcarnitines and oleoylcarnitines (C18:1 and C16:1) and various phosphatidylcholine species containing saturated fatty acids were elevated. Increased taurine levels suggested an enhanced cysteine flux through the salvage pathway whereas increased homocysteine levels appeared to be a consequence of a massive down-regulation of cystathionine β lyase (cystathionine β synthase) and a reduced flux through the transsulfuration pathway. The findings demonstrate that a suboptimal selenium status causes alterations in lipid and carbon-1 metabolism in mouse liver. These changes may contribute to the development of diseases associated with a suboptimal selenium status.     

Dictyostelium myosin II heavy-chain kinase A is activated by heparin, DNA and acidic phospholipids and inhibited by polylysine, polyarginine and histones.

Dictyostelium myosin II heavy-chain kinase A (MHCK A) is activated by autophosphorylation. Heparin and DNA, as well as vesicles composed of phosphatidylserine or phosphatidylinositol, were found to increase the initial rate of MHCK A autophosphorylation 5-10-fold in a Ca(2+)-independent manner. The negatively charged molecules also increased the activity of the autophosphorylated MHCK A by about 2-fold. In contrast, positively charged polypeptides such as poly(D-lysine), poly(L-lysine), poly(L-arginine) and histones strongly inhibited (IC50 of 0.5 micrograms/ml) the activity of the active, autophosphorylated MHCK A. Similar levels of inhibition, on a weight basis, were observed for poly(L-lysine) fractions with molecular weights from 3800 to 150,000-300,000. The inhibition was competitive with respect to peptide substrate and mixed with respect to ATP. At much higher concentrations poly(L-lysine) also inhibited the ability of MHCK A to autophosphorylate. It is proposed that negatively charged compounds and autophosphorylation increase the activity of MHCK A by weakening the interaction between the catalytic domain and a positively charged autoinhibitory domain.     

Efficient 40°C fermentation of <Emphasis Type="SmallCaps">l</Emphasis>-lysine by a new<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis> mutant developed by genome breeding

We have recently developed a new L-lysine-producing mutant of Corynebacterium glutamicum by "genome breeding" consisting of characterization and reconstitution of a mutation set essential for high-level production. The strain AHP-3 was examined for L-lysine fermentation on glucose at temperatures above 35 degrees C, at which no examples of efficient L-lysine production have been reported for this organism. We found that the strain had inherited the thermotolerance that the original coryneform bacteria was endowed with, and thereby grew and produced L-lysine efficiently up to 41 degrees C. A final titer of 85 g/l after only 28 h was achieved at temperatures around 40 degrees C, indicating the superior performance of the strain developed by genome breeding. When compared with the traditional 30 degrees C fermentation, the 40 degrees C fermentation allowed an increase in yield of about 20% with a concomitant decrease in final growth level, suggesting a significant transition of carbon flux distribution in glucose metabolism. DNA array analysis of metabolic changes between the 30 degrees C and 40 degrees C fermentations identified several differentially expressed genes in central carbon metabolism although we could not find stringent control-like global induction of amino-acid-biosynthetic genes in the 40 degrees C fermentation. Among these changes, two candidates were picked out as the potential causes of the increased production at 40 degrees C; decreased expression of the citrate synthase gene gltA and increased expression of malE, the product of which involves regeneration of pyruvate and NADPH.     

外源添加代谢中间体对产琥珀酸放线杆菌厌氧发酵制备丁二酸的影响

考察了外源添加中间代谢产物对菌体生长及发酵产酸的影响,结果表明添加0.5g/L磷酸烯醇式丙酮酸(PEP)时丁二酸产量最高。围绕产琥珀酸放线杆菌NJ113厌氧发酵产丁二酸的代谢网络进行代谢通量分析,发现添加PEP后己糖磷酸途径(HMP)与糖酵解途径(EMP)的通量比由39.4∶60.3提高至76.8∶22.6,解决了丁二酸合成过程中还原力不足的矛盾,导致PEP生成草酰乙酸的通量提高了23.8%,丁二酸代谢通量从99.8mmol/(gDCW·h)增至124.4mmol/(gDCW·h),而副产物乙酸及甲酸的代谢通量分别降低了22.9%、15.4%;关键酶活分析结果表明,添加0.5g/LPEP后PEP羧化激酶比酶活达到1910U/mg,与对照相比提高了74.7%,而丙酮酸激酶的比酶活降低了67.5%。最终丁二酸浓度为29.1g/L,收率达到76.2%,比未添加PEP时提高了11.0%。     

A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.     

Asymmetrical synthesis of L-homophenylalanine using engineered Escherichia coli aspartate aminotransferase

Site-directed mutagenesis was performed to change the substrate specificity of Escherichia coli aspartate aminotransferase (AAT). A double mutant, R292E/L18H, with a 12.9-fold increase in the specific activity toward L-lysine and 2-oxo-4-phenylbutanoic acid (OPBA) was identified. E. coli cells expressing this mutant enzyme could convert OPBA to L-homophenylalanine (L-HPA) with 97% yield and more than 99.9% ee using L-lysine as amino donor. The transamination product of L-lysine, 2-keto-6-aminocaproate, was cyclized nonenzymatically to form Delta(1)-piperideine 2-carboxylic acid in the reaction mixture. The low solubility of L-HPA and spontaneous cyclization of 2-keto-6-aminocaproate drove the reaction completely toward L-HPA production. This is the first aminotransferase process using L-lysine as inexpensive amino donor for the L-HPA production to be reported.     

A conjugate of 5-fluorouridine-poly(L-lysine) and an antibody reactive with human colon carcinoma

The ribose moiety of 5-fluorouridine (FUR) was oxidized with periodate and the product was bound through a poly(L-lysine) bridge to monoclonal antibodies, denoted SF25MAb, reactive with a human colon carcinoma LS180. The antibody was linked via its polysaccharide (previously oxidized with periodate) to the poly(L-lysine)-drug conjugate. The linking of FUR-poly(L-lysine) to the antibody markedly increased the latter's binding to the tumor cells. A relatively lower increase was also observed with conjugates of nonrelated antibodies, such as anti-hepatitis B surface antigen and anti-epidermal growth factor receptor antibodies. The pharmacological activity of the specific conjugate FUR-poly(L-lysine) -SF25MAb was higher than that of the drug-substituted polymer alone. The poly(L-lysine) bridge caused toxic effects in vivo, even though substituted both by FUR and by antibody. Therefore, the additional unreacted lysyl residues were blocked by succinylation. Partial blocking of free amino groups on the conjugate rendered it nontoxic but decreased its cell-binding capacity, though to a level still higher than that of the original unmodified antibody. The pharmacological activity of the specific conjugate after blocking was also reduced and necessitated prolonged incubation periods or higher concentrations. Following periodate oxidation and reduction, FUR was as effective as the clinically preferred compound 5-fluoro-2'-deoxyuridine in vitro and in vivo, against the LS180 colon carcinoma. Experiments in nude mice, with LS180 tumor subcutaneous xenotransplants, showed that FUR-poly(L-lysine)-SF25MAb (blocked by succinylation) was not toxic and was effective in the retardation of tumor growth.(ABSTRACT TRUNCATED AT 250 WORDS)