Participation of lysine-sensitive aspartokinase in threonine production by S-2-aminoethyl cysteine-resistant mutants of Serratia marcescens.

S-2-Aminoethyl cysteine (AEC) reduced both growth rate and final growth level of Serratia marcescens Sr41. The growth inhibition was completely reversed by lysine. AEC inhibited the activity of lysine-sensitive aspartokinase to a lesser extent than lysine. The AEC addition to the medium lowered not only the level of lysine-sensite aspartokinase but also those of homoserine dehydrogenase and threonine deaminase, whereas lysine repressed the aspartokinase alone. To select mutations releasing lysine-sensitive aspartokinase from feedback controls, AEC-resistant colonies were isolated from strains HNr31 and HNr53, both of which were previously found to excrete threonine on the minimal plates but not on the plates containing excess lysine. Two of 280 resistant colonies excreted large amounts of threonine. Strains AECr174 and AECr301, derived from strains HNr31 and HNr53, respectively, lacked both feedback inhibition and repression of lysine-sensitive aspartokinase. These strains produced about 7 mg of threonine per ml in the medium containing glucose and urea.     

Transductional construction of a threonine-producing strain of Serratia marcescens.

A threonine-producing strain of Serratia marcescens Sr41 was constructed according to the following process. Thr- strain E-60 was derived from strain HNr59 having constitutive levels of threonine-sensitive aspartokinase and homoserine dehydrogenase. Thr+ transductant T-570 was constructed from strain E-60 and phage grown on strain HNr21 having feedback-resistant threonine-sensitive aspartokinase and homoserine dehydrogenase. This transductant lacked both feedback inhibition and repression for the two enzymes. Thr- strain N-11 was derived from strain AECr174 lacking feedback inhibition and repression of lysine-sensitive aspartokinase. Subsequently, the threonine region of strain T-570 was transduced into strain N-11. One of the THR+ transductants, strain T-693, produced markedly high levels of the two aspartokinases and homoserine dehydrogenase, which were insensitive to feedback inhibition. This strain produced about 25 mg of threonine per ml in the medium containing sucrose and urea.     

Transductional construction of a threonine-producing strain of Serratia marcescens.

A threonine-producing strain of Serratia marcescens Sr41 was constructed according to the following process. Thr- strain E-60 was derived from strain HNr59 having constitutive levels of threonine-sensitive aspartokinase and homoserine dehydrogenase. Thr+ transductant T-570 was constructed from strain E-60 and phage grown on strain HNr21 having feedback-resistant threonine-sensitive aspartokinase and homoserine dehydrogenase. This transductant lacked both feedback inhibition and repression for the two enzymes. Thr- strain N-11 was derived from strain AECr174 lacking feedback inhibition and repression of lysine-sensitive aspartokinase. Subsequently, the threonine region of strain T-570 was transduced into strain N-11. One of the THR+ transductants, strain T-693, produced markedly high levels of the two aspartokinases and homoserine dehydrogenase, which were insensitive to feedback inhibition. This strain produced about 25 mg of threonine per ml in the medium containing sucrose and urea.     

Comparison of the three aspartokinase isozymes in Bacillus subtilis Marburg and 168.

The levels of two aspartokinase isozymes, a lysine-sensitive enzyme and an aspartokinase that is inhibited synergistically by lysine plus threonine, differ strikingly in different strains of Bacillus subtilis. In derivatives of B. subtilis 168 growing in minimal medium, the predominant isozyme is the lysine-sensitive aspartokinase. In B. subtilis ATCC 6051, the Marburg strain, the level of the lysine-sensitive aspartokinase is much lower during growth in minimal medium, and the major aspartokinase activity is the lysine-plus-threonine-sensitive isozyme. Molecular cloning and nucleotide sequence determination of the genes for the lysine-sensitive isozymes from the two B. subtilis strains and their upstream control regions showed these genes to be identical. Evidence that the lysine-sensitive aspartokinase, referred to as aspartokinase II, is distinct from the threonine-plus-lysine-sensitive aspartokinase comes from the observation that disruption of the aspartokinase II gene by recombinational insertion had no effect on the latter. Mutants were obtained from the aspartokinase II-negative strain that also lacked the threonine-plus-lysine-sensitive aspartokinase, which will be referred to as aspartokinase III. Aspartokinase II could be selectively restored to these mutants by transformation with plasmids carrying the aspartokinase II gene. Study of the growth properties of the various mutant strains showed that the loss of either aspartokinase II or aspartokinase III had no effect on growth in minimal medium but that the loss of both enzymes interfered with growth unless the medium was supplemented with the three major end products of the aspartate pathway. It appears, therefore, that aspartokinase I alone cannot provide adequate supplies of precursors for the synthesis of lysine, threonine, and methionine by exponentially growing cells.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

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.     

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.     

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.     

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.     

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     

Selection and Characterization of Maize Variants Resistant to S-(2-Aminoethyl)-L-Cysteine and 5-Methyltryptophan

Using tissue culture selection techniques, variants resistant to S-(2-aminoethyl)-L-cysteine (AEC) and 5-methyltryptophan(5MT) were, respectively, isolated from Opaque-2 maize inbred line “Zhongxi 037/02” and “Zhongxi 091/02”. After growing 5 months on AEC free medium, the AEC-resistant cell line (Raec) still showed high level AEC resistance which was 4 times. higher than that of its wild type, “Zhongxi 037/02”. The resistance was expressed at the plant level. New cultures initiated from shoot tissue of plants regenerated from Raec was also resistant to AEC inhibition. The free pool of lysine, threonine, isoleucine, methionine and arginine increased 0.5–3.4 fold in Raec culture. The aspartokinase from both AEC-resistant and -sen- sitive lines exhibited similar sensitivity to lysine and AEC inhibition. But the aspartokinase activity in the resistant line was 2.3 times of that in sensitive line. Seed were obtained from the plants resistant to AEC when crossed with pollen of sensitive plants. The resistance of 5MT-resistant cell line, tested after growth for 11 months on nonselection medium, was 3.5 times higher than that of its wild type, “Zhongxi 091/02”. The 5MT-resistance was possibly due to the accumulation of free tryptophan (from 0 to 61.6 nmol/g fr. wt) in the resistant cells. There was also an increase in free phenylalanine (14.5 fold) and tyrosine (28.8 fold).     

Threonine production by ethionine-resistant mutants of Serratia marcescens.

Ethionine reduced both the growth rate and the final growth level of Serratia marcescens Sr41. Growth inhibition was completely reversed by methionine. Strain D-315, defective in homoserine dehydrogenase I, was more sensitive to ethionine-mediated growth inhibition than was the wild-type strain. Ethionine-resistant mutants were isolated from cultures of strain D-316, which was derived from strain D-315 as a threonine deaminase-deficient mutant. Of 60 resistant colonies, 7 excreted threonine on minimal agar plates. One threonine-excreting strain, ETr17, was highly resistant to ethionine and, moreover, insensitive to methionine-mediated growth inhibition, whereas the parent strain was sensitive. When cultured in minimal medium with or without excess methionine, strain ETr17 had a higher homoserine dehydrogenase level than did strain D-316. The homoserine dehydrogenase activity was not inhibited by threonine or methionine. Transductional analysis revealed that the ethionine-resistant (etr-1) mutation carried by strain ETr17 was located in the metBM-argE region and caused the derepressed synthesis of homoserine dehydrogenase II. Strain ETr17 had a higher aspartokinase level than did the parent strain. By transductional cross with the argE+ marker, the etr-1 mutation was transferred into strain D-562 which was derived from D-505, a strain defective in aspartokinases I and III. The constructed strain had a higher aspartokinase level than did strain D-505 in medium with or without excess methionine, indicating that the etr-1 mutation led to the derepressed synthesis of aspartokinase II. Strain ETr17 produced about 8 mg of threonine per ml of medium containing sucrose and urea.     

Multivalent Repression of Aspartic Semialdehyde Dehydrogenase in Escherichia coli K-12

Mutants of Escherichia coli in which the lysine-sensitive aspartokinase is feedback-resistant are described. In these strains, as well as in the wild type, aspartic semialdehyde dehydrogenase is subject to multivalent repression by lysine, threonine, and methionine. When these amino acids were added to a culture in minimal medium, the differential rate of synthesis of the enzyme dropped to zero and remained there for about one generation.     

水稻抗氨基酸类似物突变体的筛选——Ⅰ.抗S-(2-氨乙基)L-半胱氨酸(AEC)突变体的筛选

以赖氨酸类似物S-(2-氨乙基)L-半胱氨酸(AEC)为选择剂,从水稻花药培养中筛选出一个抗性突变体(R_(AEC))。突变体愈伤组织经过6个月继代培养后仍保持抗性稳定。R_(AEC)再生植株根尖诱导的愈伤组织经过3个月继代培养也保持稳定的抗性。R_(AEC)细胞内赖氨酸含量提高了近2倍,苏氨酸提高5倍多。其他氨基酸,如蛋氨酸、酪氨酸、丝氨酸等都有较大量的提高。 R_(AEC)愈伤组织对赖氨酸加苏氨酸混合物也具有抗性。突变体植株较原始类型稍矮小,巳正常结实。     

Regulation of the synthesis of aspartokinase 3 of Escherichia coli by amino acids other than lysine

Summary When Escherichia coli B is grown in the presence of methionine, leucine and some other amino acids, lysine-sensitive aspartokinase (aspartokinase III) and aspartic semialdehyde dehydrogenase syntheses are derepressed. This can be explained by a synergistic inhibition between lysine and these amino acids on the lysine-sensitive aspartokinase, which leads to a decrease of the lysine intracellular pool.     

Derepression and Repression of Lysine-sensitive Aspartokinase during in Vitro Culture of Carrot Root Tissue

The increment of lysine-sensitive aspartokinase (EC 2.7.2.4) activity during in vitro culture of carrot (Daucus carota, cv. Oogata sanzun) root tissue was explained in terms of derepression caused by an earlier decrease in the endogenous level of lysine, a possible end product repressor. Tissue content of free lysine decreased to about one-third of the initial level after 1 day of culture and no lysine was detected in the 2nd day. Inclusion of lysine (0.1 to 1.0 millimolar) in the culture medium resulted in a specific suppression of increase in lysine-sensitive aspartokinase activity without affecting the increase in threonine-sensitive aspartokinase activity.     

Transductional construction of a threonine-hyperproducing strain of Serratia marcescens: lack of feedback controls of three aspartokinases and two homoserine dehydrogenases

To construct a threonine-hyperproducing strain of Serratia marcescens Sr41, the six regulatory mutations for three aspartokinases and two homoserine dehydrogenases were combined in a single strain by three transductional crosses. The constructed strain, T-1026, carried the lysC1 mutation leading to lack of feedback inhibition and repression of aspartokinase III, the thrA1(1) mutation desensitizing aspartokinase I to feedback inhibition, the thrA2(1) mutation releasing feedback inhibition of homoserine dehydrogenase I, the two hnr mutations derepressing aspartokinase I and homoserine dehydrogenase I, and the etr-1 mutation derepressing aspartokinase II and homoserine dehydrogenase II. The strain produced ca. 40 mg of threonine per ml of medium containing sucrose and urea. Furthermore, the productivity of strain T-1026 was compared with those of strains devoid of more than one of the six regulatory mutations.     

Transductional construction of a threonine-hyperproducing strain of Serratia marcescens: lack of feedback controls of three aspartokinases and two homoserine dehydrogenases.

To construct a threonine-hyperproducing strain of Serratia marcescens Sr41, the six regulatory mutations for three aspartokinases and two homoserine dehydrogenases were combined in a single strain by three transductional crosses. The constructed strain, T-1026, carried the lysC1 mutation leading to lack of feedback inhibition and repression of aspartokinase III, the thrA1(1) mutation desensitizing aspartokinase I to feedback inhibition, the thrA2(1) mutation releasing feedback inhibition of homoserine dehydrogenase I, the two hnr mutations derepressing aspartokinase I and homoserine dehydrogenase I, and the etr-1 mutation derepressing aspartokinase II and homoserine dehydrogenase II. The strain produced ca. 40 mg of threonine per ml of medium containing sucrose and urea. Furthermore, the productivity of strain T-1026 was compared with those of strains devoid of more than one of the six regulatory mutations.     

Regulation of lysine- and lysine-plus-threonine-inhibitable aspartokinases in Bacillus brevis.

Further studies on the expression of the two aspartokinase activities in Bacillus bovis are presented. Aspartokinase I (previously shown to be inhibited and repressed by lysine) was found to be repressed by diaminopimelate in the wild-type strain. However, in a mutant unable to convert diaminopimelate to lysine, starvation for lysine resulted in an increase in aspartokinase I activity. Thus, lysine itself or an immediate metabolite was the true effector of repression. Aspartokinase II (previously shown to be inhibited by lysine plus threonine) was repressed by threonine. Studies with the parent strain and auxotrophs inidicated that only threonine or an immediate metabolite of threonine was involved in this repression. Methionine and isoleucine were not effectors of any of the detected aspartokinase activities. Apart from inhibition and repression controls, a third as yet undefined regulatory mechanism operated to decrease the levels of both aspartokinases as growth declined, even in mutants in which repression control was absent. In thiosine-resistant, lysine-excreting mutants with elevated levels of aspartokinase, the increase in activity could always be attributed to one enzyme or the other, never both. The existence of separate structural genes for each aspartokinase is therefore suggested.