Regulation of aspartate-derived amino acid biosynthesis in the yeastSaccharomyces cerevisiae

The activity of three enzymes, aspartokinase, homoserine dehydrogenase, and homoserine kinase, has been studied in the industrial strainSaccharomyces cerevisiae IFI256 and in the mutants derived from it that are able to overproduce methionine and/or threonine. Most of the mutants showed alteration of the kinetic properties of the enzymes aspartokinase, which was less inhibited by threonine and increased its affinity for aspartate, and homoserine dehydrogenase and homoserine kinase, which both lost affinity for homoserine. Furthermore, they showed in vitro specific activities for aspartokinase and homoserine kinase that were higher than those of the wild type, resulting in accumulation of aspartate, homoserine, threonine, and/or methionine/S-adenosyl-methionine (Ado-Met). Together with an increase in the specific activity of both aspartokinase and homoserine kinase, there was a considerable and parallel increase in methionine and threonine concentration in the mutants. Those which produced the maximal concentration of these amino acids underwent minimal aspartokinase inhibition by threonine. This supports previous data that identify aspartokinase as the main agent in the regulation of the biosynthetic pathway of these amino acids. The homoserine kinase in the mutants showed inhibition by methionine together with a lack or a reduction of the inhibition by threonine that the wild type undergoes, which finding suggests an important role for this enzyme in methionine and threonine regulation. Finally, homoserine dehydrogenase displayed very similar specific activity in the mutants and the wild type in spite of the changes observed in amino acid concentrations; this points to a minor role for this enzyme in amino acid regulation.     

Homoserine and quorum‐sensing acyl homoserine lactones as alternative sources of threonine: a potential role for homoserine kinase in insect‐stage Trypanosoma brucei

De novo synthesis of threonine from aspartate occurs via the β‐aspartyl phosphate pathway in plants, bacteria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: homoserine kinase (HSK) and threonine synthase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK specifically phosphorylates L‐homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, homoserine, homoserine lactone and certain acyl homoserine lactones (AHLs) were found to substitute for threonine in growth media for wild‐type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinformatic studies predict that tsetse endosymbionts possess part (up to homoserine in Wigglesworthia glossinidia) or all of the β‐aspartyl phosphate pathway (Sodalis glossinidius). In addition S. glossinidius is known to produce 3‐oxohexanoylhomoserine lactone which also supports trypanosome growth. We propose that T. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.     

Isolation of auxotrophic mutants from acetobacter methanolicus MB 58

The lysine content of the biomass of the acidophilic facultatively methylotrophic bacterium Acetobacter methanolicus MB 58 was increased by genetic manipulations. A homoserine auxotroph, MB 58.196, and a threonine auxotroph, MB 58.195, were obtained from Acetobacter methanolicus MB 58 by N-methyl-N′-nitro-N-nitrosoguanidine treatment. Investigations of enzyme activities revealed that the homoserine auxotroph lacks homoserine dehydrogenase activity, and the threonine auxotroph lacks homoserine kinase activity. Concerning the lysine-producing ability, only the homoserine auxotrophic mutant accumulates lysine in the intracellular pool. The intracellular lysine content of this mutant was increased 40-fold. An excretion of amino acids into the medium was not detected. A homoserine resistant mutant, MB 58.196.10, isolated from MB 58.196 by UV-irradiation, was able to excrete lysine. About 95% of free lysine were found in the culture medium. Altogether, the free lysine concentration was increased 800-fold in comparison to the wild-type strain. By these genetic manipulations the total lysine concentration of MB 58.196 was increased to 2.7% and of MB 58.196.10 to 56% in comparison to the wild-type strain.     

Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots

Aspartokinase (EC 2.7.2.4), homoserine-dehydrogenase (EC 1.1.1.3) and dihydrodipicolinic-acid-synthase (EC 4.2.1.52) activities were examined in extracts from 1-year-old and 11-year-old cell suspension cultures and whole roots of garden carrot (Daucus carota L.). Aspartokinase activity from suspension cultures was inhibited 85% by 10 mM L-lysine and 15% by 10mM L-threonine. In contrast, aspartokinase activity from whole roots was inhibited 45% by 10 mM lysine and 55% by 10 mM threonine. This difference may be based upon alterations in the ratios of the two forms (lysine-and threonine-sensitive) of aspartokinase, since the activity is consistently inhibited 100% by lysine+threonine. Only one form each of homoserine dehydrogenase and of dihydrodipicolinic acid synthase was found in extracts from cell suspension cultures and whole roots. The regulatory properties of either enzyme were identical from the two sources. In both the direction of homoserine formation and aspartic--semialdehyde formation, homoserine dehydrogenase activities were inhibited by 10mM threonine and 10 mM L-cysteine in the presence of NADH or NADPH. KCl increased homoserine dehydrogenase activity to 185% of control values and increased the inhibitory effect of threonine. Dihydrodipicolinic acid synthase activities from both sources were inhibited over 80% by 0.5 mM lysine. Aspartokinase was less sensitive to inhibition by low concentrations of lysine and threonine than were dihydrodipicolinic acid synthase and homoserine dehydrogenase to inhibition by the respective inhibitors.     

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     

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.     

Localisation and characterisation of homoserine dehydrogenase isolated from barley and pea leaves

Two forms of homoserine dehydrogenase exist in the leaves of both barley and pea; one has a large molecular weight and is inhibited by threonine, the other is of smaller molecular weight and insensitive to threonine but inhibited by cysteine. The subcellular localisation of these enzymes has been examined. Both plants have 60–65% of the total homoserine dehydrogenase activity present in the chloroplast and this activity is inhibited by threonine. The low molecular weight, threonine-insensitive form is present in the cytoplasm. Total homoserine dehydrogenase activity from barley leaves showed progressive desensitisation towards threonine with age in a similar manner to that previously described for maize. It was shown that the effect was due to desensitisation of the chloroplast enzyme, and not to an increase in the insensitive cytoplasm enzyme. No corresponding desensitisation to threonine was detected in pea leaves. The different forms of homoserine dehydrogenase could be separated from pea leaves by chromatography on Blue Sepharose; the threonine-sensitive enzyme passed straight through and the threonine insensitive form was bound. A similar separation of the barley leaf isoenzymes was obtained using Matrex Gel Red A affinity columns; in this case however, the threonine-sensitive isoenzyme was bound. In both plants, the threonine insensitive isoenzyme was subject to greater inhibition by cysteine than was the threonine-sensitive isoenzyme.Abbreviation HSDH homoserine dehydrogenase     

Aspartate kinase and homoserine dehydrogenase ofCandida utilis

Aspartate kinase and homoserine dehydrogenase activity were assayed in a dialyzed cell-free extract ofCandida utilis. Aspartate kinase was partly inhibited by ATP-Mg and by Mg²⁺ alone. There appear to be two isoenzymes of aspartate kinase in the yeast, one heatlabile, the other relatively heat-stable. The first is subject to feedback inhibition by threonine, the other is threonine-resistant. Neither aspartate kinase nor homoserine dehydrogenase is the rate-limiting enzyme in methionine biosynthesis. Homoserine dehydrogenase measured in the forward direction showed an activity five times higher than aspartate kinase. No regulatory interaction could be demonstrated for this enzyme. No repression of aspartate kinase and homoserine dehydrogenase synthesis by threonine, methionine or both amino acids was observed.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

Amplification of three threonine biosynthesis genes inCorynebacterium glutamicum and its influence on carbon flux in different strains

Summary The hom-thrB operon (homoserine dehydrogenase/homoserine kinase) and the thrC gene (threonine synthase) of Corynebacterium glutamicum ATCC 13 032 and the hom _FBR (homoserine dehydrogenase resistant to feedback inhibition by threonine) alone as well as hom _FBR-thrB operon of C. glutamicum DM 368-3 were cloned separately and in combination in the Escherichia coli/C. glutamicum shuttle vector pEK0 and introduced into different corynebacterial strains. All recombinant strains showed 8- to 20-fold higher specific activities of homoserine dehydrogenase, homoserine kinase, and/or threonine synthase compared to the respective host. In wild-type C. glutamicum, amplification of the threonine genes did not result in secretion of threonine. In the lysine producer C. glutamicum DG 52-5 and in the lysine-plus-threonine producer C. glutamicum DM 368-3 overexpression of hom-thrB resulted in a notable shift of carbon flux from lysine to threonine whereas cloning of hom _FBR-thrB as well as of hom _FBR in C. glutamicum DM 368-3 led to a complete shift towards threonine or towards threonine and its precursor homoserine, respectively. Overexpression of thrC alone or in combination with that of hom _FBR and thrB had no effect on threonine or lysine formation in all recombinant strains tested. Offprint requests to: B. J. Eikmanns     

Mechanism of repression of methionine biosynthesis in Escherichia coli. II. The effect of metJ mutations on the free amino acid pool

Summary Ethionine-resistant mutants (metJ mutants) were isolated and characterized as constitutive in the biosynthesis of methionine. Such mutations resulted in marked differences or alterations in the free amino acid pool. In some strains the levels of threonine and histidine were elevated by as much as 13 and 22 times that of the wild type level. The possibility that structural modifications of methionyl-tRNA were giving rise to constitutive methionine biosynthesis and the apparent aberrations in the free amino acid pool, was in large part ruled out by a comparison of the mobilities of wild type and mutant methionyl-tRNA on benzoylated DEAE-cellulose columns. The results obtained are consistent with the view that the product of the metJ locus is a repressor protein which is directly involved in the repression of the methionine genes.     

Minor threonine dehydratase encoded within the threonine synthetic region of Bacillus subtilis

Challenging auxotrophs on metabolites that are precursors of a biosynthetic step involving a mutated enzyme has revealed a new class of suppressor mutations which act by derepressing a minor enzyme activity not normally detected in the wild-type strain. These indirect, partial suppressor mutations which allow isoleucine auxotrophs to grow on homoserine or threonine have been analyzed to determine their effect on enzymes involved in the biosynthesis of these amino acids. It has been found that one class of these suppressor mutations (sprA) leads to the derepression of homoserine kinase, homoserine dehydrogenase, and a minor threonine dehydratase that is not sufficiently active to be detected in the wild-type strain. The gene encoding this second threonine dehydratase activity has been found to be located between the structural genes for homoserine kinase and homoserine dehydrogenase. The results of these experiments indicate that plating of auxotrophs on precursors of a biosynthetic step involving mutated enzymes could prove to be a valuable method for the detection of regulatory mutants as well as a possible tool in studying the evolution of biochemical pathways.     

Target of serine inhibition in Escherichia coli

L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I.     

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.     

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.     

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.     

General control of amino acid biosynthesis in mutants of Candida spec. EH 15/D

The general control of amino acid biosynthesis was investigated in Candida spec. EH 15/D, using single and double mutant auxotrophic strains and prototrophic revertants starved for their required amino acids. These experiments show that starvation for lysine, histidine, arginine, leucine, threonine, proline, serine, methionine, homoserine, asparagine, glutamic acid or aspartic acid can result in derepression of enzymes. A correlation was found between the degree of derepression, growth of strains, and concentration of required amino acids. The amino acids pool pattern of mutants and revertants is different from that in the wild type strain.     

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.