Energetics of threonine uptake by pod wall tissues of Vicia faba L.

Kjeldahl assays showed that the pod wall of Vicia faba fruits behaves as a transitory reservoir of nitrogen. We have studied the properties and energetics of amino-acid uptake during the accumulating stage of pod wall development. A comparative analysis using various inhibitors or activators of the proton pump has been carried out i) on threonine uptake, ii) on the acidifying activity of the tissues, and iii) on the transmembrane potential difference of mesocarp cells. Except for the effect of dicyclohexylcarbodiimide which could not be satisfactorily explained, all other results obtained with ATPase inhibitors, uncouplers and fusicoccin were consistent with the view of a transport energized by the proton-motive force. Adding threonine to a medium containing fragments of pericarp or of endocarp induced a pH change (to-wards more alkaline values) of the medium and a membrane depolarization of the storage cells which depended on the amino-acid concentration added. These data indicate H⁺-threonine cotransport in the pod wall of broad bean. Moreover, because p-chloromercuribenzenesulphonic acid inhibits threonine uptake without affecting the transmembrane potential difference, it is concluded that the threonine carrier possesses a functional SH-group located at the external side of the plasmalemma.Abbreviations CCCP carbonylcyanide- m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - FC fusicoccin - PCMBS p-chloromercuribenzenesulphonic acid - PD potential difference     

Inactivation of Escherichia coli threonine synthase by dl-Z-2-amino-5-phosphono-3-pentenoic acid

The rhizocticines and plumbemicines are two groups of di- and tripeptid antibiotics thought to interfere with threonine or threonine-related metabolism. Z-2-amino-5-phosphono-3-pentenoic acid, the common unusual amino acid constituent of the rhizocticines and plumbemicines, was found to irreversibly inhibit Escherichia coli threonine synthase in a time-dependent reaction that followed pseudo-first order and saturation kinetics. These data provide evidence that the toxicity of the rhizocticines and plumbemicines is due to the inhibition of threonine synthase by Z-2-amino-5-phosphone-3-pentenoic acid, which is liberated by peptidases after uptake into the target cell. Additionally, methods for the purification of threonine synthase from an overproducing E. coli strain and for the enzymatic synthesis of l-homoserine phosphate are described.Abbreviations APPA Z-2-amino-5-phosphono-3-pentenoic acid - HSerP l-homoserine phosphate - PEP phosphoenolpyruvate - PLP pyndoxal 5-phosphate - TS threonine synthase     

Stabilization of threonine production in an Escherichia coli overproducing strain using tightly regulated T7 promoter system

The industrial production of compounds by E. coli strains is often accompanied with variability in yield levels. To investigate the mechanism of such instability the over-production of threonine was used as a model. The instability in this strain appears to be caused by a metabolic burden resulting in occurrence of low producing revertants. A successful application of the tightly regulated T7 expression system is presented as a possible solution providing a substantial stabilization of the threonine production.     

Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.     

Evaluation of the<Emphasis Type="Italic"> Escherichia coli</Emphasis> threonine deaminase gene as a selectable marker for plant transformation

The initial step in the synthesis of isoleucine (Ile) is the conversion of threonine to -ketobutyrate. This reaction is carried out by threonine deaminase (TD), which is feedback-regulated by Ile. Mutations in TD that manifest insensitivity to Ile feedback inhibition result in intracellular accumulation of Ile. Previous reports have shown that in planta expression of the wild-type Escherichia coli TD, ilvA, or an Ile-insensitive mutant designated ilvA-466, increased cellular concentrations of Ile. A structural analog of Ile, l-O-methylthreonine (OMT), is able to compete effectively with Ile during translation and induce cell death. It has been postulated that OMT could therefore be utilized as an effective selective agent in plant engineering studies. To test this concept, we designed two binary plasmids that harbored an nptII cassette and either the wild-type ilvA or mutant ilvA-466. The ilvA coding sequences were fused to a plastid transit peptide down stream of a modified 35S CaMV promoter. Tobacco transformations were set up implementing a selection protocol based on either kanamycin or OMT. The ilvA gene was effectively utilized as a selectable marker gene to identify tobacco transformants when coupled with OMT as the selection agent. However, the transformation efficiency was substantially lower than that observed with nptII using kanamycin as the selection agent. Moreover, in a subset of the ilvA transformants and in a majority of the ilvA-466 transgenic lines, a severe off-type was observed under greenhouse conditions that correlated with increased levels of expression of the ilvA transgene.Abbreviations ELISA enzyme-linked immunosorbent assay - Ile isoleucine - OMT l-O-methylthreonine - nptII neomycin phosphotransferase II - TD threonine deaminase     

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.     

Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea

A desensitized aspartate kinase (AK) gene has been developed as a non-antibiotic selection marker for use in the production of transgenic chickpea (Cicer arietinum L.). Transgenic shoots regenerated from embryo explants bombarded with the desensitized AK gene were selected on media containing two amino acids, lysine and threonine (LT). Approximately 15% of the putative transgenic shoots of vars. P-362 and P-1042 survived after 4 weeks of growth on MSB5 medium (MS mineral salts and B5 vitamins) containing 2 µM thidiazuron (TDZ) and 2 mM lysine and 2 mM threonine. These shoots were subsequently grown on MSB5 medium supplemented with 2 µM TDZ and 5 mM lysine and 5 mM threonine, and nearly 1% continued to grow after 16 weeks of selection. A phosphinothricin (PPT) selection system for Agrobacterium-mediated chickpea transformation was also developed. Three varieties of chickpea, P-362, P-1042 and P-1043, were successfully used for Agrobacterium transformation. Following Agrobacterium infection, 3-8% of the regenerated shoots remained green and continued to grow on MSB5 medium supplemented with 2.5 mg l^-1 PPT. Increasing the concentrations of PPT to 15 mg l^-1 reduced transgenic shoot production in P-362, P-1042 and P-1043 to 0.7%, 1.2% and 1.1%, respectively. Selected putatively transformed shoots of all three varieties were rooted and grown to maturity. Southern hybridization analysis revealed single as well as multiple integration of genes in selected transgenic lines. The level of AK activity detected in LT-selected plants was higher than that detected in the non-transformed control.Abbreviations AK: Aspartate kinase - CP: Chlorophenol red - GUS: -Glucuronidase - IBA: Indole-3-butyric acid - Kn: Kinetin (6-furfuryl aminopurine) - LT: Lysine plus threonine - MSB5: Murashige and Skoog salts with B5 vitamins - nptII: Neomycin phosphotransferase II - pat: Phosphinothricin-acetyltransferase - PPT: Phosphinothricin - TDZ: Thidiazuron [1-phenyl-3-(1,2,3-thiadiazol-5-YL) urea]Communicated by P. LakshmananJ. Sen and N. Tewari-Singh have contributed equally to this article.     

Threonine diffusion and threonine transport in Corynebacterium glutamicum and their role in threonine production

Transmembrane threonine fluxes (i.e., uptake, diffusion, and carrier-mediated excretion) all contribut-ing to threonine production by a recombinant strain of Corynebacterium glutamicum, were analyzed and quantitated. A threonine-uptake carrier that transports threonine in symport with sodium ions was identified. Under production conditions (i.e., when internal threonine is high), this uptake system catalyzed predominantly threonine/threonine exchange. Threonine export via the uptake system was excluded. Threonine efflux from the cells was shown to comprise both carrier-mediated excretion and passive diffusion. The latter process was analyzed after inhibition of all carrier-mediated fluxes. Threonine diffusion was found to proceed with a first-order rate constant of 0.003 min^–1 or 0.004 μl min^–1 (mg dry wt.)^–1, which corresponds to a permeability of 8 × 10^–10 cm s^–1. According to this permeability, less than 10% of the efflux observed under optimal conditions takes place via diffusion, and more than 90% must result from the activity of the excretion carrier. In addition, the excretion carrier was identified by (1) inhibition of its activity by amino acid modifying reagents and (2) its dependence on metabolic energy in the form of the membrane potential. Activity of the excretion system depended on the membrane potential, but not on the presence of sodium ions. Threonine export in antiport against protons is proposed. Received: 25 August 1995 / Accepted: 18 October 1995     

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987).Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability.A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of Ap^rTc^r clones after cultivation in non selective media).We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.     

Identification of a novel human MAST4 gene, a new member of human microtubule associated serine/threonine kinase family

Human protein kinases make up a large superfamily of homologous proteins, which are related by virtue of their kinase domains (also known as catalytic domains). Here, we report the cloning and characterization of a novel human MAST4 (microtubule associated serine/threonine kinase family member 4) gene, which locates on human chromosome 5q13. The MAST4 cDNA is 7587 base pairs in length and encodes a putative protein of 2435 amino acids which contains a serine/threonine kinase domain and a PDZ domain. MAST4 protein has 64, 63, 59, and 39% identical amino acid residues with MAST1, MAST2, MAST3, and MASTL, respectively. RT-PCR analysis revealed a relatively high expression level of MAST4 in most normal human tissues, with the exception of in testis, small intestine, colon, and peripheral blood leukocyte. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 808–815. The text was submitted by the authors in English. The nucleotide sequences reported in this paper have been submitted to GenBank under accession number: AY830839. These two authors contributed equally to this paper.     

In silico strategy to rationally engineer metabolite production: A case study for threonine in Escherichia coli

Genetic engineering of metabolic pathways is a standard strategy to increase the production of metabolites of economic interest. However, such flux increases could very likely lead to undesirable changes in metabolite concentrations, producing deleterious perturbations on other cellular processes. These negative effects could be avoided by implementing a balanced increase of enzyme concentrations according to the Universal Method [Kacser and Acerenza (1993) Eur J Biochem 216:361–367]. Exact application of the method usually requires modification of many reactions, which is difficult to achieve in practice. Here, improvement of threonine production via pyruvate kinase deletion in Escherichia coli is used as a case study to demonstrate a partial application of the Universal Method, which includes performing sensitivity analysis. Our analysis predicts that manipulating a few reactions is sufficient to obtain an important increase in threonine production without major perturbations of metabolite concentrations. Biotechnol. Bioeng. 2009;103: 609–620. © 2009 Wiley Periodicals, Inc.     

Production of threonine byBrevibacterium flavum containing threonine biosynthesis genes fromEscherichia coli

Genes of the threonine operon ofEscherichia coli were used for the construction of aBrevibacterium flavum strain excreting threonine. Using the shuttle vector pCEM300 and a newly constructed shuttle vector pEC71 (7.1 kb, Km^r/Nm^r), various plasmids carryingE. coli thr genes were prepared. Mutants resistant to the threonine analog 2-amino-3-hydroxyvaleric acid (AHV) were isolated after the ethyl methanesulfonate treatment ofB. flavum carrying these recombinant plasmids. A mutant ofB. flavum CCM 351 carrying the cloned genesthrA andthrB accumulated 12 g/L of threonine after 48 h of cultivation.     

Fine structure analysis of the threonine operon in Escherichia coli K-12.

Summary A fine structure analysis of the threonine operon in Escherichia coli K-12 was performed by deletion mapping. Lambda transducing bacteriophages carrying various parts of the threonine operon were isolated from strains in which the lacZ gene was fused to a thr gene. We tested for recombination between deletions of the threonine promotor extending into the threonine operon, carried by the phage, and bacterial thr auxotrophs. The relative order of thrO (operator) mutations was established. We propose that an operator region is located between a promoter region and the structural genes. Mutations leading to the desensitization of the aspartokinase I-homoserine dehydrogenase I towards threonine were localized in two different regions of the thrA gene.     

Tissue culture isolation of a second mutant locus for increased threonine accumulation in maize

Summary Regenerable maize (Zea mays L.) tissue cultures were selected for ability to grow in the presence of inhibitory (1.0–1.5 mM) concentrations of L-lysine plus L-threonine. Testcross kernels from one regenerated plant (LT20) segregated for wild-type and high free threonine concentration in a 11 ratio consistent with a single dominant gene for high free threonine. Free threonine concentrations (nmol/mg dry weight) increased an average of 29-fold in bulked F₂ kernel samples from heterozygous mutant plants, and the total (free plus protein-bound) threonine concentration increased 68%. Increases in protein-bound methionine, lysine and glycine concentrations were also noted, suggesting a possible effect of the mutation on protein concentration and composition. Allelism tests with a previously selected mutant line, Ltr ^*19, showed that two unlinked, codominant genes conditioned the high free threonine phenotype. Based on a separate study of aspartate kinase feedback inhibition characteristics in the two mutant lines, we propose that the mutant alleles [gene and allele designations are according to guidelines for maize genetic nomenclature (Burnham et al. 1975)] be designated Ask-LT19 and Ask2-LT20 for the Ltr ^*19 and LT20 mutants, respectively.     

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.     

Resistance to serine in Bacillus subtilis: identification of the serine transporter YbeC and of a metabolic network that links serine and threonine metabolism

The Gram-positive bacterium Bacillus subtilis uses serine not only as a building block for proteins but also as an important precursor in many anabolic reactions. Moreover, a lack of serine results in the initiation of biofilm formation. However, excess serine inhibits the growth of B. subtilis. To unravel the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine concentrations by three targeted and non-targeted genome-wide screens. All screens as well as genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. The screen for serine-resistant mutants also identified mutations that result in increased serine degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine toxicity results from interference with threonine biosynthesis.     

Allele-specific suppression of aSaccharomyces cerevisiae prp20 mutation by overexpression of a nuclear serine/threonine protein kinase

The yeast PRP20 protein is homologous to the RCC1 protein of higher eukaryotes and is required for mRNA export and maintenance of nuclear structure. RCC1/PRP20 act as guanine nucleotide exchange factors for the nuclear Ras-like Ran/GSP1 proteins. In a search forprp20-10 allele-specific high-copy-number suppressors, theKSP1 locus, encoding a serine/threonine protein kinase was isolated. Ksp1p is a nuclear protein that is not essential for vegetative growth of yeast. Inactivation of the kinase activity by a mutation affecting the catalytic center of the Ksp1p eliminated the suppressing activity. Based on the isolation of a protein kinase as a high-copy-number suppressor, the phosphorylation of Prp20p was examined. In vivo labeling experiments showed that Prp20p is a phosphoprotein; however, deletion of the KSP1 kinase did not affect Prp20p phosphorylation.     

Characterization of DNA sequences encoding a novel isoform of the 55 kDa B regulatory subunit of the type 2A protein serine/threonine phosphatase of Arabidopsis thaliana

We have indentified a novel gene (AtB) encoding a previously uncharacterized isoform of the B regulatory subunit of the type 2A serine/threonine protein phosphatase (PP2A) of Arabidopsis, and show that mRNA derived from the AtB gene accumulates in all Arabidopsis organs. In addition, we examined the expression of the three genes encoding the A regulatory subunit of Arabidopsis PP2A and show these genes are expressed in all organs as well. Taken together, our results suggest a myriad of PP2A subunit combinations, possibly with distinct substrate specificities, may occur within each Arabidopsis cell.     

Toxicity and accumulation of thallium in bacteria and yeast

Thallium sulphate inhibited microbial growth, withBacillus megaterium KM, more sensitive to the metal thanSaccharomyces cerevisiae andEscherichia coli. Inhibition ofB. megaterium KM andS. cerevisiae, but not ofE. coli, was alleviated by increasing the potassium concentration of the medium; inhibition of respiration ofS. cerevisiae, but not ofE. coli, was similarly alleviated. Thallium was rapidly bound, presumably to cell surfaces, byS. cerevisiae andE. coli, and was progressively accumulated by energy-dependent transport systems (probably concerned primarily with potassium uptake) with both organisms. Thallium uptake kinetics suggested more than one transport system operated in yeast, possibly reflecting a multiplicity of potassium transport systems. ApparentK _m andK _i values for competitive inhibition of thallium uptake by potassium indicatedS. cerevisiae to have a higher affinity for thallium uptake than for potassium, whileE. coli had a transport system with a higher affinity for potassium than for thallium. The likely systems for thallium transport are discussed. A mutant ofE. coli with tenfold decreased sensitivity to thallium was isolated and apparently effected surface binding of thallium in amounts equivalent to the wild type organism, but showed no subsequent uptake and accumulation of the metal from buffer, even though it was able to accumulate potassium to normal intracellular concentrations during growth. Abbreviations: Metal are referred to by their recognised atomic symbols (e.g. TI = Thallium; K = potassium; Co = cobalt)