Characteristics of lysine transport in a wild type strain and lysine-producing mutant of Corynebacterium glutamicum

An active transport system high specific for 1-lysine was found in the cells of the wild strain of Corynebacterium glutamicum, Km being about 10 microM. Accumulation of lysine was higher, if the cells were cultivated on a medium containing glucose. The cells of the homoserine-deficient lysine producer have no alterations in the lysine transport. The lysine transport was also studied in three lysine producing analog resistant mutants (two mutants are resistant to aminoethylcysteine and one to lysine hydroxamate). The key enzyme of the lysine biosynthesis, aspartate kinase, is insensitive to the feedback inhibition by the mixture of lysine and threonine in all the mutant studied; at the same time the cells of these mutants grown on a glucose-containing medium above mentioned alterations are suggested to provide the resistance to the lysine analog.     

Selection and Characterization of Asparagus Mutants Resistant to Inhibition by Lysine Plus Threonine

Asparagus officinalis calli were induced from shoot of seedlings. After mutagenization, two lysine plus threonine resistant mutant lines (LTR2, LTR3) were obtained by selectionnonselection-reselection procedures with 2 mmol/1 lysine plus threonine. LTR2 and LTR3 caIli remained resistance to lysine plus threonine after being subcultured for 1 year, and both of them showed cross resistance to 1 mmol/l aminoethylcysteine. In resistant calli, the free lysine, methionine and an unknown amino acid were l-l0 times more than those in controls.     

Threonine accumulation in the seeds of a barley mutant with an altered aspartate kinase

Barley (Hordeum vulgare L.) mutants altered in the regulation of synthesis of aspartate-derived amino acids were sought by screening embryos for growth on a medium containing lysine plus threonine. One mutant, Rothamsted 2501, was selected with good growth. From the segregation of resistance in the following generations, it was concluded that the resistance was conferred by a dominant gene, Lt1. No homozygous Lt1/Lt1 fertile plants have been recovered. Partially purified aspartate kinase preparations from resistant and sensitive plants were separated on DEAE-cellulose chromatography into three peaks of activity (I, II, III) and the feedback regulatory properties of these peaks determined. These peaks are considered to be three isozymic forms of aspartate kinase, one predominantly sensitive to threonine and two sensitive to lysine or lysine plus S-adenosyl methionine. The feedback characteristics of one of the peaks of aspartate kinase activity from resistant plants were changed such that lysine was half-maximally inhibitory at 10 rather than 0.4mm. Increases in the concentrations of the free pools of threonine (4×) and methionine (2×) were measured in young plants grown on a basal medium. Threonine in the soluble fraction of mature seeds from resistant plants was increased from 0.8 to 9.6% of the total threonine content. The total content of both threonine and methionine of the seeds was increased by 6% compared with grain of similar nitrogen content.S.E.R. acknowledges the receipt of a Council of Europe Scholarship through The British Council. Part of this was also supported by EEC Grant 473.     

Genetic and amino-acid analysis of two maize threonine-overproducing,lysine-insensitive aspartate kinase mutants

The aspartate-derived amino-acid pathway leads to the production of the essential amino-acids lysine, methionine, threonine and isoleucine. Aspartate kinase (AK) is the first enzyme in this pathway and exists in isoforms that are feedback inhibited by lysine and threonine. Two maize (Zea mays L.) threonine-overproducing, lysine-insensitive AK mutants (Ask1-LT19 and Ask2-LT20) were previously isolated. The present study was conducted to determine the map location of Ask2 and to examine the amino-acid profiles of the Ask mutants. The threonine-overproducing trait conferred by Ask2-LT20 was mapped to the long arm of chromosome 2. Both mutants exhibited increased free threonine concentrations (nmol/mg dry weight) over wild-type. The percent free threonine increased from approximately 2% in wild-type kernels to 37–54% of the total free amino-acid pool in homozygous mutant kernels. Free methionine concentrations also increased significantly in homozygous mutants. Free lysine concentrations were increased but to a much lesser extent than threonine or methionine. In contrast to previous studies, free aspartate concentrations were observed to decrease, indicating a possible limiting factor in threonine synthesis. Total (free plus protein-bound) amino-acid analyses demonstrated a consistent, significant increase in threonine, methionine and lysine concentrations in the homozygous mutants. Significant increases in protein-bound (total minus free) threonine, methionine and lysine were observed in the Ask mutants, indicating adequate protein sinks to incorporate the increased free amino-acid concentrations. Total amino-acid contents (nmol/kernel) were approximately the same for mutant and wild-type kernels. In five inbred lines both Ask mutations conferred the threonine-overproducing phenotype, indicating high expressivity in different genetic backgrounds. These analyses are discussed in the context of the regulation of the aspartate-derived amino-acid pathway.     

L-lysine production at 65°C by auxotrophic-regulatory mutants ofBacillus stearothermophilus

Summary The amino acid L-lysine was produced from auxotrophic-regulatory mutants ofBacillus stearothermophilus at a temperature of 60–65°C. One of the mutants (AEC 12 A5, S-(2-aminoethyl)-cysteine^r, homoserine^–), produced L-lysine at the concentration of 7.5 g/l in shaken flasks in minimal medium containing 5% glucose. Culture conditions for optimizing L-lysine production were not investigated. The aspartokinase activity of the wild strainB. stearothermophilus Zu 183 was inhibited by lysine alone and by threonine plus lysine. AEC resistant mutants showed an aspartokinase activity genetically desensitized to the feedback inhibition. Optimal temperature and pH of aspartokinase were 45°C and 9.5, respectively. The data provide significant evidence that mutants of the speciesB. stearothermophilus have a potential value for amino acid production.     

A Mutant of Arabidopsis thaliana lpar;L.) Heynh. with Modified Control of Aspartate Kinase by Threonine

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 (ak1and ak2) were found to be unlinked.     

beta-lactam antibiotic production by Streptomyces clavuligerus mutants impaired in regulation of aspartokinase

This work describes isolation and characterization of Streptomyces clavuligerus mutants resistant to the lysine analogue S-(2-aminoethyl)-L-cysteine (AEC). The mutation to AEC resistance was shown to affect the feedback regulation of aspartokinase; 70% of the mutants isolated had aspartokinase activity insensitive to concerted feedback inhibition by lysine plus threonine. Among these mutants, 70% (about 50% of the total AEC-resistant strains isolated) showed significant overproduction of beta-lactam antibiotics.     

Homoserine and threonine pools of borrelidin resistant Saccharomyces cerevisiae mutants with an altered aspartokinase

Borrelidin is a specific inhibitor of the threonyl-tRNA-synthethase. A class of dominant borrelidin resistant mutants (BOR1) of Saccharomyces cerevisiae was biochemically characterized. The mutants possess an altered aspartokinase which is insensitive to threonine inhibition. The threonine and homoserine pools in these mutants are 22 times larger than in the wild type. By feeding aspartate under a variety of conditions the homoserine pool is increased even 57 times whilst the threonine pool is reduced.     

Synthesis of 2-(d-Arabino-tetra-hydroxybutyl)pyrazine-5-sulfonic Acid

An efficient method for the isolation of dihydrodipicolinate synthase (DPS)-defective threonine producers from a Br evibacterium strain with feedback-sensitive aspartokinase (AK, Ak^s) was established. After mutagenesis of a strain with AK, No. 70, mutants resistant to α-amino-β- hydroxyvaleric acid were isolated and then selected as to threonine productivity in the presence of diaminopimelic acid. DPS activity in the strains in which the threonine production was inhibited by lysine was found to be absent or reduced to less than 10 % of the level in the parent. On the other hand, the strains in which the production was not inhibited by lysine were conventional threonine producers with feedback-resistant homoserine dehydrogenases (HDs and HD^Rs) and wild type DPS. The HD activities of most of the threonine mutants were also markedly reduced. However, only one mutant lacking DPS, DK330, exhibited an HD level comparable to that in the parent and produced the largest amount of threonine among the threonine producers obtained. The formation of HD and HK in strain DK330 was hardly repressed by the addition of methionine. Under the optimum conditions, strain DK330 produced 12.4 g/1 of threonine, while a typical HD type threonine producer, BK29, produced 9.9 g/1.     

Aspartokinase of a lysine producing mutant ofMicrococcus glutamicus

Aspartokinase fromMicrococcus glutamicus AEC RN-13-6/1 [a homoserine requiring, S-(2-aminoethyl)-L-cysteine resistant, lysine producing strain] was purified 71 fold. The partially purified enzyme was inhibited by L-lysine. L-threonine, L-methionine, L-isoleucine, L-valine and L-phenylalanine activated the enzyme and reversed the inhibition by L-lysine. Aspartokinase activity was not derepressed by growth-limiting concentrations of L-threonine and/or L-methionine. It was not repressed by an excess of L-lysine (20 mM) and/or L-isoleucine (15.3 mM). The degree of activation or inhibition by amino acids was dependant on the composition of the growth medium. This observation is in contrast with the enzyme from the original (non-lysine-producing) strain which was inhibited by lysine or threonine and in a concerted manner by threonine plus lysine.     

Selection and characterization of a feedback-insensitive tissue culture of maize

Tissue culture selection techniques were used to isolate a maize (Zea mays L.) variant D33, in which the aspartate family pathway was less sensitive to feedback inhibition by lysine. D33 was recovered by successively subculturing cultures originally derived from immature embryos on MS medium containing growth-inhibitory levels of lysine+threonine. The ability of D33 to grow vigorously on lysine+ threonine medium was retained after growth for 12 months on nonselection medium. New cultures initiated from shoot tissues of plants regenerated from D33 also were resistant to lysine+threonine inhibition. The K_i of aspartokinase for its feedback inhibitor, lysine, was about 9-fold higher in D33 than for the enzyme from unselected cultures. The free pools of lysine, threonine, isoleucine and methionine were increased 2–9-fold in D33 cultures. This was consistent with the observed change in feedback regulation of aspartokinase, the first enzyme common to the biosynthesis of these amino acids in the aspartate pathway. The accumulated evidence including the stability of resistance in the cultures, the resistance of cultures initiated from regenerated plants, the altered feedback regulation, and the increased free amino acids, indicates a mutational origin for these traits in line D33.Abbreviation LT lysine+threonine in equimolar concentration Paper No. 10880, Scientific Journal Series, Minnesota Agricultural Expertment Station     

Effect of Pyruvate Kinase Deficiency on l-Lysine Productivities of Mutants with Feedback-resistant Aspartokinases

The cultural conditions were investigated for a Brevibacterium flavum mutant, No. 2–190, with a low level of citrate synthase (CS) and with feedback-resistant phosphoenoipyruvate (PEP) carboxylase and aspartokinase (AK). The productivity was increased from 28 to 38 g/1 (as the HC1 salt) with a medium containing 10% glucose. From this strain, pyruvate kinase (PK)-defective mutants were derived and selected as to the inability to grow on ribose. Among them, strain Kl-18 showed higher lysine productivity than the parent under all cultural conditions tested, and produced 43 g/1 of lysine, at maximum. A lysine-producing mutant, No. 536–4, with a feedback- resistant AK was derived from PK-defective strain KH-21 which had low CS activity and a feedback-resistant PEP carboxylase. The mutant was isolated by a new selection method, that is, on the basis of resistance to α-amino-ß-hydroxyvaleric acid, a threonine analogue plus lysine. In this strain, HD had been altered so as to become feedback-resistant at the same time, resulting in the byproduction of threonine and isoleucine. The total amount of these aspartate family amino acids was higher on molar basis than that of lysine produced by strain No. 2–190.     

Mitochondrial cytochrome b genes with a six-nucleotide deletion or single-nucleotide substitutions confer resistance to antimycin A in the yeast Kluyveromyces lactis

Extrachromosomal mutants resistant to antimycin, from the yeast Kluyveromyces lactis, have been isolated, genetically characterized, and assigned to two specific genetic loci (Brunner et al., 1987). In the present work the cytochrome b nucleotide sequence from six of these mutants was determined. Five mutants had single point mutations, corresponding to transversions. In one mutant, a six-base-pair deletion, beginning at nucleotide 689, was observed. The amino acid sequence derived from the coding strand showed that, in three independent antimycin-resistant mutants, a change of asparagine 31 into lysine took place (two of these mutants are also resistant to diuron). Two other mutants showed a change from lysine 228 into isoleucine (or methionine). Leucine 230, isoleucine 231, and threonine 232, were lost in the deletion mutant and were replaced by serine.     

Segregation for endosperm lysine in F2, F3 and F4 progeny from a cross of in vitro-selected and unselected cultivar of rice

Summary Lysine is a limiting amino acid for optimal nutritional quality in rice grain. In vitro selections using inhibitory levels of lysine plus threonine or s-aminoethylcysteine allow the predictable recovery of variants with elevated levels of lysine and protein. These methods may generate useful starting germplasm for plant breeders. This study was conducted to define the genetics of lysine mutants in progeny from crosses of mutants derived from cells cultured in vitro in the presence of inhibitory levels of lysine plus threonine and s-(2-aminoethyl)-cysteine. In vitro selections produce a wide range of mutants, including endosperm mutants with elevated lysine and protein levels as well as mutants for high and low seed weights. Mutants were analyzed for lysine content by the endosperm half-seed method in which the halves without the embryo were ground and acid hydrolyzed for amino acid determinations. The halves with the embryos were preserved for later germination. In two different F₂ populations derived from a cross of a selected mutant x M-101, a parental marker, there was an inverse relationship between seed weight and percent lysine in endosperm protein (R² 0.52 and 0.56). The F₂ segregation patterns show that elevated lysine is inherited as a recessive gene and that increased lysine is correlated with decreased seed size. F₃ and F₄ data provide evidence for the transmission of high lysine genes to advanced germplasm in rice. This work supports our earlier conclusions that high lysine phenotypes can be recovered predictably from in vitro selections. The elevated lysine phenotypes are frequently, but not exclusively, associated with opaque seed. Some segregants from crosses produced increased lysine in plants with near normal seed weight and good fertility.Research done under the auspices of the USDA, ARS, Plant Sciences Institute, Plant Molecular Biology Laboratory, Beltsville, MD 20705, USA     

Lysine and threonine metabolism are subject to complex patterns of regulation in Arabidopsis

To study the regulation of lysine and threonine metabolism in plants, we have transformed Arabidopsis thaliana with chimeric genes encoding the two bacterial enzymes dihydrodipicolinate synthase (DHPS) and aspartate kinase (AK). These bacterial enzymes are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Transgenic plants expressing the bacterial DHPS overproduced lysine, but lysine levels were quite variable within and between transgenic genotypes and there was no direct correlation between the levels of free lysine and the activity of DHPS. The most lysine-overproducing plants also exhibited abnormal phenotypes. However, these phenotypes were detected only at early stages of plant growth, while at later stages, new buds emerged that looked completely normal and set seeds. Wild-type plants exhibited relatively high levels of free threonine, suggesting that in Arabidopsis AK regulation may be more relaxed than in other plants. This was also supported by the fact that expression of the bacterial AK did not cause any dramatic elevation in this amino acid. Yet, the relaxed regulation of threonine synthesis in Arabidopsis was not simply due to a reduced sensitivity of the endogenous AK to feedback inhibition by lysine and threonine because growth of wild-type plants, but not of transgenic plants expressing the bacterial AK, was arrested in media containing these two amino acids. The present results, combined with previous studies from our laboratory, suggest that the regulation of lysine and threonine metabolism is highly variable among plant species and is subject to complex biochemical, physiological and environmental controls. The suitability of these transgenic Arabidopsis plants for molecular and genetic dissection of lysine and threonine metabolism is also discussed.     

Control of the aspartokinase ofStreptomyces noursei and its AEC-resistant mutants

Summary Addition of L-lysine to cultures ofS. noursei enhanced the production of nourseothricin. The aspartokinase of the wild-type strain was under concerted feedback inhibition by lysine plus threonine but was stimulated by lysine alone. Threonine in the medium increased the synthesis of enzyme. 10% of the mutants resistant to AEC showed a higher specific production of the antibiotic.     

The effect of aspartate-derived amino acids (lysine,threonine, methionine) on the growth of excised embryos of wheat and barley

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     

Cloning of a DNA fragment fromCorynebacterium glutamicum conferring aminoethyl cysteine resistance and feedback resistance to aspartokinase

Summary TheCorynebacterium glutamicum/Escherichia coli shuttle vector plasmid pZ1 was used to clone the S-(2-aminoethyl)-d,l-cysteine (AEC)-resistance gene from a lysine-excreting, AEC-resistant strain ofC. glutamicum, the aspartokinase activity of which was released from feedback inhibition by mixtures of lysine and threonine or AEC and threonine respectively. A recombinant plasmid designated pCS2 carrying a 9.9-kb chromosomal insert that conferred AEC resistance and the ability to excrete lysine to its host was isolated. The aspartokinase activity of the pCS2-carrying strain was resistant towards inhibition by mixtures of lysine and threonine or AEC and threonine respectively. By deletion analysis the DNA region conferring AEC resistance to the host and feedback resistance to its aspartokinase activity could be confined to a 1.2-kb DNA fragment.     

Lysine and threonine biosynthesis in sorghum seeds: characterisation of aspartate kinase and homoserine dehydrogenase isoenzymes

Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion‐exchange chromatography [diethylaminoethyl (DEAE)‐Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S‐200), one peak of AK activity co‐eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine‐sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2‐aminoethylether)‐N,N,N′N′‐tetraacetic acid, calmodulin, S‐adenosylmethionine (SAM), S‐2‐aminoethyl‐l ‐cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine‐sensitive isoenzyme and a second predominant threonine‐insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.     

Biochemical studies with bi-resistant mutants (ethambutol plus streptomycin and isoniazid plus streptomycin) ofMycobacterium smegmatis ATCC 607

Biochemical characteristics of bi-resistant mutants (resistant to ethambutol plus streptomycin or isoniazid plus streptomycin) of mycobacteria isolated by replica plating fromMycobacterium smegmatis ATCC were compared with those of the drug-susceptible strains. Reduced incorporation of [¹⁴C]uracil, [³H]lysine and [¹⁴C]acetate into RNA, protein and phospholipids respectively was seen in the resistant mutants. Total phosphorlipids were enhanced in ethambutol plus streptomycin resistant mutant and decreased in isoniazid plus streptomycin resistant mutant. There were similar changes in levels of individual phospholipids. The resistant mutants revealed an accumulation of phospholipids in the cell wall, and a marked decrease of phospholipids in the cell membrane in comparison to the susceptible strain. Several qualitative alterations in the polypeptide profile (with respect to number and molecular weight) of the crude protein extract and of different subcellular compartments were seen in the resistant mutants.