Molecular cloning and expression of s-(2-aminoethyl)-L-cysteine resistant aspartokinase gene ofCorynebacterium glutamicum

Summary A 2.9 kb DNA fragment encoding s-(2-aminoethyl)-l-cysteine (AEC) resistant aspartokinase ofCorynebacterium glutamicum was cloned on theC. glutamicum/E. coli shuttle vector pECCG117. A recombinant plasmid, designated pAK12, conferred the AEC resistance, the ability to excrete lysine and threonine, and the 3–5 fold increased specific activity of aspartokinase to host strain.     

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.     

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.     

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.     

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.     

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.     

Effect of different levels of aspartokinase of the lysine production by Corynebacterium lactofermentum

A 2.9-kb SacI fragment containing the ask-asd operon, encoding aspartokinase and aspartate-semialdehyde dehydrogenase, was cloned from an aminoethylcysteine-resistant, lysine-producing Corynebacterium lactofermentum strain. Enzymatic analysis showed that the aspartokinase (ASK) activity was completely resistant to inhibition by mixtures of lysine and threonine. Comparison of the deduced amino acid sequence of the submit of the ask gene showed three amino acid residue changes with ask genes encoding wild-type, feedback-sensitive enzymes. Three C. lactofermentum strains, one being aspartokinase-negative, one carrying two ask genes on the chromosome and one having a sixfold higher specific ASK activity than the parental strain, were constructed by transconjugation and electroporation, and used to analyse the role of ASK in the lysine production by C. lactofermentum. The results indicate that, in this study, feed-back-resistant ASK is necessary for high-level lysine production, but dispensable for lysine and diaminopimelate synthesis required for cell growth.     

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).     

Production of l-Lysine by Leucine Auxotrophs Derived from AEC Resistant Mutant of Brevibacterium lactofermentum

The effect of amino acids was examined on the production of l-lysine by AEC resistant mutant of B. lactofermentum. Among amino acids tested, only leucine showed strong specific inhibition. In order to release the production of l-lysine from this negative effect of leucine, leucine auxotrophs were derived from AEC resistant strain of B. lactofermentum. Most of these leucine auxotrophs produced larger amount of l-lysine (maximally 41 mg/ml) than the parental strain which produced about 18 mg/ml of l-lysine. It was confirmed that leucine auxotrophs derived from AEC resistant mutant of other glutamate producing bacteria, B. saccharolyticum and Corynebacterium glutamicum. These results suggested that leucine might directly or indirectly affect the biosynthesis of lysine.

However, this increase in lysine productivity of leucine auxotrophs could not be explained by the alteration of aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3). These enzymes are key enzymes in lysine and threonine biosynthesis, respectively.     

Regulation of lysine and dipicolinic acid biosynthesis in Bacillus brevis ATCC 10068: significance of derepression of the enzymes during the change from vegetative growth to sporulation

Lysine biosynthetic pathway enzymes of Bacillus brevis ATCC 1068 were studied as a function of stage of development (growth and sporulation). The synthesis of aspartic-2-eemialdehyde dehydrogenase (ASA-dehydrogenase), dihydrodipicolinate synthase (DHDPA-synthase), DHPA-reductase and diaminopimelate decarboxylase (DAP-decarboxylase) was found not to be co-regulated, since lysine was not a co-repressor for these enzymes. Unlike the aspartokinase isoenzymes, the other enzymes of the lysine pathway were not derepressed in thiosine-resistant, lysine-excreting mutants. Thus, the aspartokinase isoenzymes were the key enzymes during growth and regulation of lysine biosynthesis through restriction of l-ASA synthesis via feedback control by lysine on the aspartokinases was therefore suggested.In contrast to other Bacillus species, the levels of the lysine biosynthetic pathway enzymes of strain ATCC 10068 were not derepressed during the change from vegetative growth to sporulation. Two control mechanisms, enabling the observed preferential channelling of carbon for the synthesis of spore-specific diaminopimelic acid (DAP) and dipicolinic acid (DPA) were a) loss of DAP-decarboxylase, b) inhibition of DHDPA-reductase by DPA. Increase in the level of the DAP pool during sporulation, as a consequence of the loss of DAP-decarboxylase, and its relevance to the non-enzymatic formation of DPA has been discussed.Abbreviations l-ASA l-aspartic-2-semialdehyde - DAP diaminopimelic acid - DPA dipicolinic acid - DHDPA dihydrodipicolinate - AGM aspargine-glycerol medium - PY peptone-yeast extract - NB+NSM nutrient broth plus nutrient sporulation medium     

Aspartokinase genes lysC alpha and lysC beta overlap and are adjacent to the aspartate beta-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum

Summary A 2.1 kb DNA fragment of the recombinant plasmid pCS2, isolated from an aminoethyl cysteine (AEC)-resistant and lysine-producing Corynebacterium glutamicum mutant strain, and which confers AEC resistance and lysine production on the wild-type G. glutamicum ATCC 13032 was analysed. DNA sequence analysis of this fragment revealed three large open reading frames (ORFs). The incomplete ORF1 does not contain the 5 end of the coding region. ORF2, which uses the same reading frame as ORF1, is identical to the 3 end of ORF1 and encodes a putative protein of 172 amino acids (aa) and of M_r 18 584. ORF3 encodes a putative protein of 344 as and of M_r 36275. The amino acid sequences deduced from ORF1 and ORF2 display strong homologies to those of the - and -subunits of the Bacillus subtilis aspartokinase II. It is therefore proposed that the incomplete ORF1, termed lysC, encodes part of the -subunit of the C. glutamicum aspartokinase whereas the complete ORF2, termed lysC, encodes the -subunit of the same enzyme. ORF2 is responsible for AEC resistance and lysine production due to a feedback-resistant aspartokinase. The amino acid sequence deduced from ORF3, termed asd, is highly homologous to that of the Streptococcus mutans aspartate -semialdehyde dehydrogenase (ASD). Plasmids carrying the C. glutamicum asd gene complemented Escherichia coli asd mutants. Increase in ASD activity by a factor of 30–60 was measured for C. glutamicum cells harbouring high copy-number plasmids with the C. glutamicum asd gene.     

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.     

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。     

Thialysine-Resistant Mutant of SALMONELLA TYPHIMURIUM with a Lesion in the thrA Gene

A mutant of Salmonella typhimurium was selected for its spontaneous resistance to the lysine analog, thialysine (S-2-aminoethyl cysteine). This strain, JB585, exhibits a number of pleiotropic properties including a partial growth requirement for threonine, resistance to thiaisoleucine and azaleucine, excretion of lysine and valine, and inhibition of growth by methionine. Genetic studies show that these properties are caused by a single mutation in the thrA gene which encodes the threonine-controlled aspartokinase-homoserine dehydrogenase activities. Enzyme assays demonstrated that the aspartokinase activity is unstable and the threonine-controlled homoserine dehydrogenase activity absent in extracts prepared from the mutant. These results explain the growth inhibition by methionine because the remaining homoserine dehydrogenase isoenzyme would be repressed by methionine, causing a limitation for threonine. The partial growth requirement for threonine during growth in glucose minimal medium may also, by producing an isoleucine limitation, cause derepression of the isoleucine-valine enzymes and provide an explanation for both the valine excretion, and azaleucine and thiaisoleucine resistance. The overproduction of lysine may confer the thialysine resistance.     

Aspartokinase of Lemna paucicostata Hegelm. 6746

A sensitive and specific method was developed for assay of aspartokinase (EC 2.7.2.4) in crude extracts of Lemna paucicostata. Lysine inhibited approximately 93%, and threonine approximately 6%; together, these amino acids inhibited 99%. Inhibition by lysine was synergistically increased by S-adenosylmethionine, which by itself had no effect on activity. Essentially complete inhibition of threonine-resistant activity was obtained with lysine, and of lysine-resistant activity with threonine. Inhibition by lysine and threonine was additive, with no indication of concerted inhibition. Aspartate concentration had no effect on the relative proportions of lysine- and threonine-sensitive activities. Aspartokinase activity was in large excess of that reported by other workers, the maximum capacity (V_max) far exceeding the in vivo requirements. Estimations of rates of aspartokinase in vivo suggest that the step catalyzed by this enzyme may not be the overall `rate-limiting' one for entry of 4-carbon units into the aspartate family of amino acids, and that feedback inhibition of this enzyme by lysine and threonine may not be a major factor in regulating flux through this step.     

Regulatory Structure of the Biosynthetic Pathway for the Aspartate Family of Amino Acids in Lemna paucicostata Hegelm. 6746, with Special Reference to the Role of Aspartokinase

Comprehensive studies were made with Lemna paucicostata Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme alone being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [¹⁴C]threonine and [¹⁴C]homoserine in Lemna. This finding excludes not only aspartokinases as an important regulatory determinant of threonine synthesis, but also two other enzymes (homoserine dehydrogenase and threonine synthase) suggested to fulfill this role. Complete inhibition of threonine synthesis was observed only in the combined presence of accumulated threonine and lysine. The physiological significance of this single example of apparent regulation of flux at the aspartokinase step, albeit under unusually stringent conditions of aspartokinase inhibition, remains to be determined. (c) Isoleucine strongly inhibits its own synthesis, probably at threonine dehydratase, without causing compensatory reduction in threonine synthesis. A fundamentally changed scheme for regulation of synthesis of the aspartate family of amino acids is presented that has important implications for improvement of the nutritional contents of these amino acids in plants.     

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.     

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.     

Mutational analysis of the feedback sites of lysine-sensitive aspartokinase of Escherichia coli

In Escherichia coli, thrA, metLM, and lysC encode aspartokinase isozymes that show feedback inhibition by threonine, methionine, and lysine, respectively. In vitro chemical mutagenesis of the cloned lysC gene was used to identify residues and regions of the polypeptide essential for feedback inhibition by lysine. The isolated lysine-insensitive mutants were demonstrated to have missense mutations in amino acid residues 323-352, and at position 250 of aspartokinase III.