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1.
Lysine is one of the nutritionally limiting amino acids in food and feed products made from maize ( Zea mays L.). Two enzymes in the lysine biosynthesis pathway, aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS), have primary roles in regulating the level of lysine accumulation in plant cells because both enzymes are feedback-inhibited by lysine. An isolated cDNA clone for maize DHPS was modified to encode a DHPS much less sensitive to lysine inhibition. The altered DHPS cDNA was transformed into maize cell suspension cultures to determine the effect on DHPS activity and lysine accumulation. Partially purified DHPS (wildtype plus mutant) from transformed cultures was less sensitive to lysine inhibition than wild-type DHPS from nontransformed cultures. Transformed cultures had cellular free lysine levels as much as four times higher than those of nontransformed controls. Thus, we have shown that reducing the feedback inhibition of DHPS by lysine can lead to increased lysine accumulation in maize cells. Increasing the capacity for lysine synthesis may be an important step in improving the nutritional quality of food and feed products made from maize. 相似文献
2.
Lysine added to grain mashes under nitrogen-limiting conditions (as in most industrial fermentations) inhibited growth of Saccharomyces cerevisiae. This inhibition was relieved by raising the assimilable nitrogen content. Lysine-induced inhibition is not mediated through accumulation of -oxoadipic acid, an intermediate of lysine metabolism which accumulates by a back up of intermediates in de novo synthesis. Lysine degradation is regulated by the synthesis of L-lysine -aminotransferase, an enzyme that catalyses the first step in one of three possible routes of lysine degradation (not previously reported in S. cerevisiae). Synthesis is repressed under nitrogenlimiting conditions, but derepressed when excess assimilable nitrogen is available. Derepression results in degradation of lysine and decreases inhibitory effects on growth. The toxic compound appears to be lysine itself.The authors are with the Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 0W0. Canada 相似文献
3.
A major nutritional drawback of many crop plants is their low content of several essential amino acids, particularly lysine. The biosynthesis of lysine in plants is regulated by several feedback loops. Dihydrodipicolinate synthase (DHPS) from Escherichia coli, a key enzyme in lysine biosynthesis, which is considerably less sensitive to lysine accumulation than the endogenous plant enzyme has been expressed in chloroplasts of tobacco leaves. Expression of the bacterial enzyme was accompanied by a significant increase in the level of free lysine. No increase in protein-bound lysine was evident. Free lysine accumulation was positively correlated with the level of DHPS activity in various transgenic plants. Compartmentalization of DHPS in the chloroplast was essential for its participation in lysine biosynthesis as no lysine overproduction was obtained in transgenic plants that expressed the bacterial enzyme in the cytoplasm. The elevated level of free lysine in the transgenic plants was sufficient to inhibit, in vivo, a second key enzyme in lysine biosynthesis, namely, aspartate kinase, with no apparent influence on lysine accumulation. The present report not only provides a better understanding of the regulation of lysine biosynthesis in higher plants but also offers a new strategy to improve the production of this essential amino acid. 相似文献
4.
Excised wheat ( Triticum aestivum L. var. Maris Freeman) and barley ( Hordeum vulgare L. var. Maris Mink) embryos were grown on medium containing both nitrate and ammonium ions. Addition of lysine (1 mM) plus threonine (1 mM) caused a synergistic inhibition of growth measured by length of first leaf or dry weight. The inhibition was specifically relieved by methionine, homocysteine and homoserine. Threonine at 0.2–0.3 mM caused half-maximal inhibition of growth at all lysine concentrations whereas lysine increased the synergistic inhibition up to 3 mM. The inhibition is explained by a model in which lysine acts as a feedback inhibitor of aspartate kinase and threonine of homoserine dehydrogenase. This is compatible with published studies of the enzymes involved. The implications of these findings for using lysine plus threonine as a selection system for lysine-overproducing cereals are discussed.Abbreviations Lys
Lysine
- Thr
Threonine
- Met
Methionine
- Hser
Homoserine
- Hcys
Homocysteine 相似文献
5.
From a screening of a few Ustilaginales fungi for free lysine accumulation, Sphacelotheca sorghi ITCC No. 591 was found to accumulate 50 μg/ml of free lysine in the culture medium. However, it was later seen that the free lysine content of the culture broths could not be increased much unless cell lysis took place.Experiments were also carried out to increase the free lysine content of the culture broths of U. maydis PRL No. 1092. It was found that U. maydis maintained on sterilised corn, yields up to 400 μg of free- l-lysine/ml in its culture medium in five days. 相似文献
6.
α,ε-Diaminopimelic acid (DAP)-requiring mutants isolated from Aerobacter aerogenes ATCC 8308 and Serratia marcescens ATCC 19180 were found to accumulate N-succinyl- l-diaminopimelic acid (SDAP) which was an intermediate in the biosynthesis of lysine in Escherichia coli. SDAP was isolated from the culture broth and identified by the behavior in paper chromatography, melting point, elementary analysis, infrared spectrum, and optical rotation.The culture conditions for SDAP production by A. aerogenes KY 7049 (DAP ?) and S. marcescens KY 8921 (DAP ?/Lys ?) were investigated. A. aerogenes KY 7049 has an absolute requirement for DAP together with a relative requirement for l-lysine. High levels of DAP (2000~4000 μg/ml) were proved to be favorable for SDAP accumulation, while if lysine along with DAP was added to the fermentation medium, optimal level of DAP for SDAP production was relatively low (about 200 μg/ml at 200 μg/ml of lysine). A variety of compounds which may conceivably affect the course of a fermentation process, i.e., carbon source, inorganic nitrogen source, amino acids, vitamines, precursors, were screened at optimal levels of lysine and DAP. Thus, the amount of SDAP accumulation reached a level of 19.9 mg/ml with the medium containing 10% glucose and 2000 μg/ml of DAP. S. marcescens KY 8921 requires either DAP or lysine for growth. Optimal level of DAP and lysine for SDAP accumulation was 50~100μg/ml. 相似文献
7.
By applying a mutagenesis/selection procedure to obtain resistance to a lysine analog, S-(2-aminoethyl)l -cysteine (AEC), a lysine overproducing mutant in Nicotiana sylvestris was isolated. Amino acid analyses performed throughout plant development and of different organs of the N. sylvestris RAEC-1 mutant, revealed a developmental-dependent accumulation of free lysine. Lysine biosynthesis in the RAEC-1 mutant was enhanced due to a lysine feedback-desensitized dihydrodipicolinate synthase (DHDPS). Several molecular approaches were undertaken to identify the nucleotide change in the dhdps-r1 gene, the mutated gene coding for the lysine-desensitized enzyme. The enzyme was purified from wild-type plants for amino end microsequencing and 10 amino acids were identified. Using dicotyledon dhdps probes, a genomic fragment was cloned from an enriched library of DNA from the homozygote RAEC-1 mutant plant. A dhdps cDNA, putatively full-length, was isolated from a tobacco cDNA library. Nucleotide sequence analyses confirmed the presence of the previously identified amino end preceded by a chloroplast transit peptide sequence. Nucleotide sequence comparisons, enzymatic and immunological analyses revealed that the tobacco cDNA corresponds to a normal type of DHDPS, lysine feedback-regulated, and the genomic fragment to the mutated DHDPS, insensitive to lysine inhibition. Functional complementation of a DHDPS-deficient Escherichia coli strain was used as an expression system. Reconstruction between the cDNA and genomic fragment led to the production of a cDNA producing an insensitive form of DHDPS. Amino acid sequence comparisons pointed out, at position 104 from the first amino acid of the mature protein, the substitution of Asn to lleu which corresponds to a dinucleotide mutation. This change is unique to the dhdps-r1 gene when compared with the wild-type sequence. The identification of the nucleotide and amino acid change of the lysine-desensitized DHDPS from RAEC-1 plant opens new perspectives for the improvement of the nutritional value of crops and possibly to develop a new plant selectable marker. 相似文献
9.
In Brevibacterium flavum, prephenate dehydratase in the phenylalanine specific biosynthetic pathway was strongly inhibited by phenylalanine and activated by tyrosine. Furthermore. the inhibition by phenylalanine was completely reversed by tyrosine. Inhibition by tyrosine of prephenate dehydrogenase in the tyrosine specific pathway was very weak. Overall regulation mechanism of the aromatic amino acid biosynthesis in B. flavum was proposed on the bases of these results and the previous findings on 3-deoxy- D- arabino-heptulosonate-7- phosphate synthetase(DAHP synthetase*) of the common pathway and on anthranilate synthetase of the tryptophan specific pathway. Two types of m-fluorophenylalanine( mFP) resistant mutants which accumulated phenylalanine alone or both phenylalanine and tyrosine, respectively, were derived. The accumulation in the former mutants was inhibited by tyrosine, but that in the latter was affected neither by tyrosine nor by phenylalanine. DAHP synthetase of the latter mutants had been desensitized from the synergistic feedback inhibition by tyrosine and phenylalanine, while prephenate dehydratase of the former mutants had been desensitized in the feedback inhibition by phenylalanine. Tyrosine auxotroph accumulated phenylalanine under tyrosine limitation and its accumulation was inhibited by the excessive addition of tyrosine. Phenylalanine auxotroph accumulated tyrosine under phenylalanine limitation and its accumulation was inhibited by the excessive addition of phenylalanine. These results in vivo strongly supported the proposed regulation mechanism in which synthesis of phenylalanine in preference to tyrosine was assumed. 相似文献
10.
Aspartate kinase (AK; EC 2.7.2.A) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids
in plants. Aspartate kinase was purified from wildtype and two maize ( Zea mays L.) genotypes carrying unlinked dominant mutations, Ask LT19 and Ask2 -LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to
characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived
amino acids in maize. Kernels of the homozygous Ask2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine,
compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 μM l-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygous Ask and homozygous Ask2 mutants required 25 and 760 μM lysine for 50% inhibition, respectively, indicating that Ask and Ask2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the
homozygous mutant Ask2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutant Ask2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site
to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor of Ask2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive
inhibition of wildtype AK. Reduced lysine sensitivity of the Ask2 gene product appeared to reduce the lysine inhibition of all of the AK activity detected in homozygous Ask2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from
the Ask and Ask2 structural genes.
Scientific paper No. 17419, Minnesota Agricultural Experiment Station projects No. 0302-4813-56 and No. 0302-4818-32
This research was supported in part by the U.S. Depatment of Agriculture Competitive Research Grants Office grant 86-CRCR-1-2019.
The authors are grateful to Charles Grissom for providing the computer programs in an IBM-PC format. 相似文献
11.
Rice cv. Sekiguchi‐asahi was identified as a lesion mimic mutant from cv. Asahi. This mutant showed light‐enhanced resistance to Magnaporthe grisea infection, inducing Sekiguchi lesion ( sl) formation and tryptamine accumulation. To elucidate the role of chloroplasts on the activation of tryptamine pathway, the effect of some photosynthetic inhibitors on light‐dependant Sekiguchi lesion formation, tryptamine accumulation, and enzyme activities was investigated. When detached leaf blades which had been pretreated with Linuron, 1, 10‐Phenanthroline or Poly‐l ‐lysine were inoculated with M. grisea, respectively, Sekiguchi lesion formation and typtamine accumulation were suppressed even under light, suppressing gene expression of tryptophan decarboxylase and monoamine oxidase activity. On the other hand, catalase activity was significantly inhibited in control leaves infected with M. grisea under light, but kept at high level in chemical‐pretreated leaves. These results suggested that photosynthetic activity in chloroplasts was involved in inhibition of antioxidant sysytem and activation of tryptamine pathway responsible for light‐enhanced resistance in sl mutant to M. grisea infection. 相似文献
12.
SYNOPSIS. Three-day-old cultures of Y and MR strains of Trypanosoma cruzi had a higher rate of lysine and arginine uptake than 10-day cultures. Amino acid uptake by cells of the MR strain was consistently higher than that of the Y strain. Flagellates separated on DEAE-cellulose columns have normal structure, motility, and infectivity; they have higher rates of lysine and arginine uptake than the original 3- and 10-day cultures. In addition, passage through DEAE-cellulose columns modified the kinetic behavior of amino acid transport systems in the flagellate membranes. Methionine inhibited uncompetitively uptake of lysine and arginine by MR and Y strains. Lysine inhibited arginine uptake by both strains by an uncompetitive mechanism. Lysine, however, inhibited the uptake of arginine by 10-day culture cells of the Y strain by a mixed-type of inhibition. Arginine also inhibited the lysine uptake of both strains by an uncompetitive mechanism. In all experiments, beyond a certain level, a further increase in inhibitor concentration resulted in a decreased inhibition, which eventually disappeared altogether. Inhibition of amino-acid uptake by any of the substances tested was not observed after passage of flagellates through a DEAE-cellulose column. A model for amino acid transport was formulated which includes a recognition site amenable to modulation by effectors. 相似文献
14.
Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grow on 2 m M lysine has been used to isolate mutants that may contain an aspartate kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an aspartate kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of aspartate kinase, were crossed to assess the effects of the simultaneous presence of these different aspartate kinase forms. A double mutant (RLT40 × RL4) was isolated and characterized by two feedback-desensitized isozymes of aspartate kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double aspartate kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1 and ak2) were found to be unlinked. 相似文献
15.
Camelina sativa (camelina) is emerging as an alternative oilseed crop due to its short growing cycle, low input requirements, adaptability to less favorable growing environments and a seed oil profile suitable for biofuel and industrial applications. Camelina meal and oil are also registered for use in animal and fish feeds; however, like meals derived from most cereals and oilseeds, it is deficient in certain essential amino acids, such as lysine. In higher plants, the reaction catalyzed by dihydrodipicolinate synthase (DHDPS) is the first committed step in the biosynthesis of lysine and is subject to regulation by lysine through feedback inhibition. Here, we report enhancement of lysine content in C. sativa seed via expression of a feedback inhibition-insensitive form of DHDPS from Corynebacterium glutamicums (CgDHDPS). Two genes encoding C. sativa DHDPS were identified and the endogenous enzyme is partially insensitive to lysine inhibition. Site-directed mutagenesis was used to examine the impact of alterations, alone and in combination, present in lysine-desensitized DHDPS isoforms from Arabidopsis thaliana DHDPS (W53R), Nicotiana tabacum (N80I) and Zea mays (E84K) on C. sativa DHDPS lysine sensitivity. When introduced alone, each of the alterations decreased sensitivity to lysine; however, enzyme specific activity was also affected. There was evidence of molecular or structural interplay between residues within the C. sativa DHDPS allosteric site as coupling of the W53R mutation with the N80V mutation decreased lysine sensitivity of the latter, but not to the level with the W53R mutation alone. Furthermore, the activity and lysine sensitivity of the triple mutant (W53R/N80V/E84T) was similar to the W53R mutation alone or the C. glutamicum DHDPS. The most active and most lysine-insensitive C. sativa DHDPS variant (W53R) was not inhibited by free lysine up to 1 mM, comparable to the C. glutamicums enzyme. Seed lysine content increased 13.6 -22.6% in CgDHDPS transgenic lines and 7.6–13.2% in the mCsDHDPS lines. The high lysine-accumulating lines from this work may be used to produce superior quality animal feed with improved essential amino acid profile. 相似文献
16.
The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analysed the importance of protein lysine methylation for plants to cope with cadmium. We analysed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species, Arabidopsis thaliana and A. lyrata, and in three populations of A. halleri with contrasting cadmium accumulation and tolerance traits. We showed that some proteins are differentially methylated at lysine residues in response to Cd and that a few genes coding KMTs are regulated by cadmium. Also, we showed that 9 out of 23 A. thaliana mutants disrupted in KMT genes have a tolerance to cadmium that is significantly different from that of wild-type seedlings. We further characterized two of these mutants, one was knocked out in the calmodulin lysine methyltransferase gene and displayed increased tolerance to cadmium, and the other was interrupted in a KMT gene of unknown function and showed a decreased capacity to cope with cadmium. Together, our results showed that lysine methylation of non-histone proteins is impacted by cadmium and that several methylation events are important for modulating the response of Arabidopsis plants to cadmium stress. 相似文献
17.
The exploitation of methoxylated aromatic monomers by Eubacterium limosum was restricted to the cleavage and consumption of the methoxyl substituents: the phenolic residues were not further attacked.
Growth characteristics were similar to those previously described for this organism on methanol. Degradation of aromatics
containing more than one methoxyl group occurred in a sequential manner and transient accumulation of intermediates (particularly
methyl-gallate) took place, though the enzymic mechanism for this phenomenon remains obscure. Degradation of 3,4,5-trimethoxybenzoate
necessitated the initial attack of the para-methoxyl group before those groups in meta positions could be metabolised. 相似文献
18.
In higher plants the essential amino acids lysine, threonine, methionine and isoleucine are synthesised through a branched
pathway starting from aspartate. The key enzyme of lysine biosynthesis in this pathway—dihydrodipicolinate synthase (DHDPS)—is
feedback-inhibited by lysine. The dhdps-r1 gene from a mutant Nicotiana sylvestris, which encodes a DHDPS enzyme insensitive to feedback inhibition, was used to improve the lysine content in pigeonpea seeds.
The dhdps-r1 coding region driven by a phaseolin or an Arabidopsis 2S2 promoter was successfully overexpressed in the seeds of pigeonpea
by using Agrobacterium transformation and particle bombardment. In 11 lines analysed, a 2- to 6-fold enhanced DHDPS activity in immature seeds at
a late stage of maturation was found in comparison to wild type. The overexpression of dhdps-r1 led to an enhanced content of free lysine in the seeds of pigeonpea from 1.6 to 8.5 times compared with wild type. However,
this was not reflected in an increase in total seed lysine content. This might be explained by a temporal discrepancy between
maximal expression of dhdps-r1 and the rate of amino acid incorporation into storage proteins. Assays of the lysine degradative enzyme lysine-ketoglutarate
reductase in these seeds showed no co-ordinated regulation of lysine biosynthesis and catabolism during seed maturation. All
transgenic plants were fertile and produced morphologically normal seeds. 相似文献
19.
In prokaryotes and plants the synthesis of the essential amino acids lysine and threonine is predominantly regulated by feed-back inhibition of aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). In order to modify the flux through the aspartate family pathway in barley and enhance the accumulation of the corresponding amino acids, we have generated transgenic barley plants that constitutively express mutant Escherichia coli genes encoding lysine feed-back insensitive forms of AK and DHPS. As a result, leaves of primary transformants (T 0) exhibited a 14-fold increase of free lysine and an 8-fold increase in free methionine. In mature seeds of the DHPS transgenics, there was a 2-fold increase in free lysine, arginine and asparagine and a 50% reduction in free proline, while no changes were observed in the seeds of the two AK transgenic lines analysed. When compared to that of control seeds, no differences were observed in the composition of total amino acids. The introduced genes were inherited in the T 1 generation where enzymic activities revealed a 2.3-fold increase of AK activity and a 4.0–9.5-fold increase for DHPS. T 1 seeds of DHPS transformants showed the same changes in free amino acids as observed in T 0 seeds. It is concluded that the aspartate family pathway may be genetically engineered by the introduction of genes coding for feed-back-insensitive enzymes, preferentially giving elevated levels of lysine and methionine. 相似文献
20.
Coprothermobacter platensis is an anaerobic, proteolytic, thermophilic bacterium, which is phylogenetically related to the genera Fervidobacterium and Thermotoga. The organism was found to reduce thiosulfate to sulfide during growth on carbohydrates and proteinaceous substrates. Growth on glucose was inhibited by hydrogen, but this inhibition was overcome by thiosulfate reduction, stirring, increasing the headspace volume and coculturing with a hydrogen-consuming methanogen. Alanine was detected during glucose fermentation, its formation was influenced by the hydrogen concentration in the gas phase suggesting an electron sink mechanism, as was previously reported for the phylogenetically related Thermotogales and the archaeal hyperthermophile Pyrococcus furiosus. 相似文献
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