Homoserine Kinase Research Articles

Molecular cloning of thehom—thrC—thrB cluster fromBacillus sp. ULM1: Expression of thethrC gene inEscherichia coli and corynebacteria, and evolutionary relationships of the threonine genes

A 6.5 kb DNA fragment containing the gene (thrC) encoding threonine synthase, the last enzyme of the threonine biosynthetic pathway, has been cloned from the DNA of Bacillus sp. ULM1 by complementation of Escherichia coli and Brevibacterium lactofermentum thrC auxotrophs. Complementation studies showed that the thrB gene (encoding homoserine kinase) is found downstream from the thrC gene, and analysis of nucleotide sequences indicated that the hom gene (encoding homoserine dehydrogenase) is located upstream of the thrC gene. The organization of this cluster of genes is similar to the Bacillus subtilis threonine operon (hom-thrC-thrB). An 1.9 kb BclI fragment from the Bacillus sp. ULM1 DNA insert 351 amino acids was found corresponding to a protein of 37462 Da. The thrC gene showed a low G + C content (39.4%) and the encoded threonine synthase is very similar to the B. subtilis enzyme. Expression of the 1.9 kb BcI DNA fragment in E. coli minicells resulted in the formation of a 37 kDa protein. The upstream region of this gene shows promoter activity in E. coli but not in corynebacteria. A peptide sequence, including a lysine that is known to bind the pyridoxal phosphate cofactor, is conserved in all threonine synthase sequences and also in the threonine and serine dehydratase genes. Amino acid comparison of nine threonine synthases revealed evolutionary relationships between different groups of bacteria.

The biosynthesis of threonine by mammalian cells: expression of a complete bacterial biosynthetic pathway in an animal cell

The coding regions for the Escherichia coli gene for aspartokinase I/homoserine dehydrogenase I (thrA) and the Corynebacterium glutamicum gene for aspartic semialdehyde dehydrogenase (asd) have been subcloned into a Simian Virus 40 (SV40)-based mammalian expression vector. Both enzyme activities are expressed in mouse 3T3 cells after transfer of the corresponding chimaeric gene. The kinetic parameters are similar to those of the native bacterial enzymes, and aspartokinase I/homoserine dehydrogenase I retains its allosteric regulation by threonine. An extract of the cells expressing aspartokinase I/homoserine dehydrogenase I, mixed with one from cells expressing aspartic semialdehyde dehydrogenase, produced homoserine when the mixture was incubated with aspartic acid, ATP and NADPH. The thrA and asd expression cassettes were combined into a single plasmid which, when transfected into 3T3 cells, enabled them to produce homoserine from aspartic acid. Homoserine-producing 3T3 cells were transfected with the plasmid pSVthrB/C (homoserine kinase and threonine synthase) and selected for growth on homoserine. Cell lines isolated from these cells expressed the complete bacterial threonine pathway, were independent of threonine for growth and could be maintained in medium which contained no free threonine. The threonine in the proteins of these cells became enriched in 15N when the culture medium contained [15N]aspartic acid. The production of homoserine and the growth of cells was at a maximum when there was more than 2.5 mM aspartate in the medium. Below this concentration the high Km of aspartokinase limited the flux through the pathway. In the presence of additional aspartic acid the new pathway could sustain a cell cycle time close to that of the same cells cultured in threonine-containing medium.

Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer.

Overproduction of isoleucine, an essential amino acid, was achieved by amplification of the gene encoding threonine dehydratase, the first enzyme in the threonine to isoleucine pathway, in a Corynebacterium lactofermentum threonine producer. Threonine overproduction was previously achieved with C. lactofermentum ATCC 21799, a lysine-hyperproducing strain, by introduction of plasmid pGC42 containing the Corynebacterium homdr and thrB genes (encoding homoserine dehydrogenase and homoserine kinase respectively) under separate promoters. The pGC42 derivative, pGC77, also contains ilvA, which encodes threonine dehydratase. In a shake-flask fermentation, strain 21799(pGC77) produced 15 g/l isoleucine, along with small amounts of lysine and glycine. A molar carbon balance indicates that most of the carbon previously converted to threonine, lysine, glycine and isolecine was incorporated into isoleucine by the new strain. Thus, in our system, simple overexpression of wild-type ilvA sufficed to overcome the effects of feedback inhibition of threonine dehydratase by the end-product, isoleucine.

Metabolic design in the amino-acid-producing bacteriumCorynebacterium glutamicum

The Gram-positive bacteriumCorynebacterium glutamicum is used for the industrial production of amino acids,e.g. ofl-glutamate andl-lysine. By cloning and expressing the various genes of thel-lysine pathway inC. glutamicum we could demonstrate that an increase of the flux ofl-4-aspartaldehydate tol-lysine could be obtained in strains with increased dihydro-dipicolinate synthase activity. Recently we detected that inC. glutamicum two pathways exist for the synthesis ofdl-2,6-diaminopimelate andl-lysine. Mutants defective in one pathway are still able to synthesize enoughl-lysine for growth but thel-lysine secretion is reduced to 50–70%. Using NMR-spectroscopy we could calculate how much of thel-lysine secreted into the medium is synthesizedvia the one and the other pathway. Amplification of the feedback-inhibition-insensitive-homoserine dehydrogenase and homoserine kinase in a highl-lysine-overproducing strain made it possible to channell of the carbon flow from the intermediate 4-aspartaldehydate toward homoserine, resulting in a high accumulation ofl-threonine. For a further flux froml-threonine tol-isoleucine the allosteric control of threonine dehydratase was eliminated.

Metabolic design in amino acid producing bacterium Corynebacterium glutamicum

The Gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of l-glutamate and l-lysine. In the last 10 years, genetic engineering and amplification of relevant structural genes have become fascinating methods for the construction of strains with desired genotypes. By cloning and expressing the various genes of the l-lysine pathway in C. glutamicum we could demonstrate that an increase of the flux of l-aspartate semialdehyde to l-lysine could be obtained in strains with increased dehydrodipicolinate synthase activity. By combined overexpression of deregulated aspartate kinase and dihydrodipicolinate synthase, the l-lysine secretion could be increased (10–20%). Recently we detected that in C. glutamicum two pathways exist for the synthesis of dl-diaminopimelate and l-lysine. Mutants defective in one pathway are still able to synthesize enough l-lysine for growth, but the l-lysine secretion is reduced to 50–70%. Using NMR spectroscopy, we could calculate how much of the l-lysine secreted into the medium is synthesized via each pathway. Amplification of the feedback inhibition-insensitive homoserine dehydrogenase and homoserine kinase in a high l-lysine overproducing strain enabled channelling of the carbon flow from the intermediate aspartate semialdehyde towards homoserine, resulting in a high accumulation of l-threonine. For a further flux from l-threonine to l-isoleucine the allosteric control of threonine dehydratase must be eliminated. In addition to all steps considered so far to be important for amino acid overproduction, the secretion into the culture medium also has to be noted. Recently it could be demonstrated that l-glutamate, l-lysine and l-isoleucine are not secreted via passive diffusion but via specific active carrier systems. Analysis of lysine-overproducing C. glutamicum strains indicates that this secretion carrier has a strong influence on the overproduction of this amino acid. Thus, for the construction of strong amino acid overproducing strains by using the gene cloning techniques, the overexpression of the genes for the export systems also seems necessary.

Metabolic design in amino acid producing bacteriumCorynebacterium glutamicum

The Gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of l-glutamate and l-lysine. In the last 10 years, genetic engineering and amplification of relevant structural genes have become fascinating methods for the construction of strains with desired genotypes. By cloning and expressing the various genes of the l-lysine pathway in C. glutamicum we could demonstrate that an increase of the flux of l-aspartate semialdehyde to l-lysine could be obtained in strains with increased dehydrodipicolinate synthase activity. By combined overexpression of deregulated aspartate kinase and dihydrodipicolinate synthase, the l-lysine secretion could be increased (10–20%). Recently we detected that in C. glutamicum two pathways exist for the synthesis of dl-diaminopimelate and l-lysine. Mutants defective in one pathway are still able to synthesize enough l-lysine for growth, but the l-lysine secretion is reduced to 50–70%. Using NMR spectroscopy, we could calculate how much of the l-lysine secreted into the medium is synthesized via each pathway. Amplification of the feedback inhibition-insensitive homoserine dehydrogenase and homoserine kinase in a high l-lysine overproducing strain enabled channelling of the carbon flow from the intermediate aspartate semialdehyde towards homoserine, resulting in a high accumulation of l-threonine. For a further flux from l-threonine to l-isoleucine the allosteric control of threonine dehydratase must be eliminated. In addition to all steps considered so far to be important for amino acid overproduction, the secretion into the culture medium also has to be noted. Recently it could be demonstrated that l-glutamate, l-lysine and l-isoleucine are not secreted via passive diffusion but via specific active carrier systems. Analysis of lysine-overproducing C. glutamicum strains indicates that this secretion carrier has a strong influence on the overproduction of this amino acid. Thus, for the construction of strong amino acid overproducing strains by using the gene cloning techniques, the overexpression of the genes for the export systems also seems necessary.

Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799.

Amplification of the operon homdr-thrB encoding a feedback-insensitive homoserine dehydrogenase and a wild-type homoserine kinase in a Corynebacterium lactofermentum lysine-producing strain resulted in both homoserine and threonine accumulation, with some residual lysine production. A plasmid enabling separate transcriptional control of each gene was constructed to determine the effect of various enzyme activity ratios on metabolite accumulation. By increasing the activity of homoserine kinase relative to homoserine dehydrogenase activity, homoserine accumulation in the medium was essentially eliminated and the final threonine titer was increased by about 120%. Furthermore, a fortuitous result of the cloning strategy was an unexplained increase in homoserine dehydrogenase activity. This resulted in a further decrease in lysine production along with a concomitant increase in threonine accumulation.

Transcriptional analysis and regulatory signals of the hom-thrB cluster of Brevibacterium lactofermentum

Two genes, hom (encoding homoserine dehydrogenase) and thrB (encoding homoserine kinase), of the threonine biosynthetic pathway are clustered in the chromosome of Brevibacterium lactofermentum in the order 5' hom-thrB 3', separated by only 10 bp. The Brevibacterium thrB gene is expressed in Escherichia coli, in Brevibacterium lactofermentum, and in Corynebacterium glutamicum and complements auxotrophs of all three organisms deficient in homoserine kinase, whereas the Brevibacterium hom gene did not complement two different E. coli auxotrophs lacking homoserine dehydrogenase. However, complementation was obtained when the homoserine dehydrogenase was expressed as a fusion protein in E. coli. Northern (RNA) analysis showed that the hom-thrB cluster is transcribed, giving two different transcripts of 2.5 and 1.1 kb. The 2.5-kb transcript corresponds to the entire cluster hom-thrB (i.e., they form a bicistronic operon), and the short transcript (1.1 kb) originates from the thrB gene. The promoter in front of hom and the hom-internal promoter in front of thrB were subcloned in promoter-probe vectors of E. coli and corynebacteria. The thrB promoter is efficiently recognized both in E. coli and corynebacteria, whereas the hom promoter is functional in corynebacteria but not in E. coli. The transcription start points of both promoters have been identified by primer extension and S1 mapping analysis. The thrB promoter was located in an 87-bp fragment that overlaps with the end of the hom gene. A functional transcriptional terminator located downstream from the cluster was subcloned in terminator-probe vectors.

Effects of the amplification of the genes coding for the L-threonine biosynthetic enzymes on the L-threonine production from methanol by a gram-negative obligate methylotroph, Methylobacillus glycogenes

We constructed recombinant plasmids carrying the genes coding for the L-threonine biosynthetic enzymes, the hom gene, the hom-thrC genes, and the thrB genes, of a gram-negative obligate methylotroph, Methylobacillus glycogenes, and examined the effects of them on the production of L-threonine from methanol. The hom gene, which encodes the homoserine dehydrogenase, and the hom-thrC genes, containing the gene coding for threonine synthase together with the hom gene, were cloned from a wild-type strain, and the thrB gene encoding the desensitized homoserine kinase was cloned from an L-threonine-producing mutant, ATR80. The recombinant plasmids were transferred into ATR80 and its L-isoleucine auxotroph, A513, by conjugation. Amplification of the genes coding for the L-threonine biosynthetic enzymes elevated the activities of the L-threonine biosynthetic enzymes of the transconjugants 10- to 30-fold over those of the strains containing only vectors. The L-threonine production from methanol in test-tube cultivation was increased about 30% and 40% by the amplification of the hom gene and the hom-thrC gene respectively, and it was slightly increased by that of the thrB gene. The effects of gene amplification were confirmed by the cultivation in 5-1 jar fermentors. The best producer, an A513 transconjugant containing the plasmid carrying the hom-thrC genes, produced 16.3 g/l L-threonine for 72 h.

Threonine synthesis from homoserine as a selectable marker in mammalian cells.

The plasmid pSVthrBC expresses the Escherichia coli thrB (homoserine kinase) and thrC (threonine synthase) genes in mouse cells and enables them to synthesize threonine from homoserine. After transfection with pSVthrBC and culture in medium containing homoserine, only cells that have incorporated pSVthrBC survive. Homoserine at concentrations greater than 1 mM is toxic to mammalian cells. Mouse cells selected from medium containing 5 mM homoserine had incorporated 20-100 copies of the plasmid per cell and had homoserine kinase activities of 0.001-0.012 nmol/min per mg of protein per copy. Cells selected from medium containing 10 mM homoserine had incorporated one or two copies of the plasmid per cell and had homoserine kinase activities of 0.06-0.39 nmol/min per mg of protein per copy. By using high concentrations of homoserine, it is possible to use pSVthrBC to select and isolate cell lines that have one or two copies of the plasmid incorporated into an active region of chromatin. CHO and HeLa cells have also been successfully transfected with pSVthrBC. COS-7 cells are naturally resistant to homoserine as they are able to metabolize homoserine.

Stable Expression of hom-1-thrB in Corynebacterium glutamicum and Its Effect on the Carbon Flux to Threonine and Related Amino Acids

The hom-1-thrB operon encodes homoserine dehydrogenase resistant to feedback inhibition by L-threonine and homoserine kinase. Stable expression of this operon has not yet been attained in different Corynebacterium glutamicum strains. We studied the use of chromosomal integration and of a low-copy-number vector for moderate expression of the hom-1-thrB operon to enable an analysis of the physiological consequences of its expression in C. glutamicum. Strains carrying one, two, or three copies of hom-1-thrB were obtained. They showed proportionally increased enzyme activity of feedback-resistant homoserine dehydrogenase and of homoserine kinase. This phenotype was stably maintained in all recombinants for more than 70 generations. In a lysine-producing C. glutamicum strain which does not produce any threonine, expression of one copy of hom-1-thrB resulted in the secretion of 39 mM threonine. Additional copies resulted in a higher, although not proportional, accumulation of threonine (up to 69 mM). This indicates further limitations of threonine production. As the copy number of hom-1-thrB increased, increasing amounts of homoserine (up to 23 mM) and isoleucine (up to 34 mM) were secreted. Determination of the cytosolic concentration of the respective amino acids revealed an increase of intracellular threonine from 9 to 100 mM and of intracellular homoserine from 4 to 74 mM as the copy number of hom-1-thrB increased. These results suggest that threonine production with C. glutamicum is limited by the efflux system for this amino acid. Furthermore, the results show the successful use of moderate and stable hom-1-thrB expression for directing the carbon flux from aspartate to threonine.

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.

The expression of Escherichia coli threonine synthase and the production of threonine from homoserine in mouse 3T3 cells.

We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.

Purification to homogeneity and characterization of homoserine kinase from wheat germ

Homoserine kinase (EC 2.7.1.39) has been purified from wheat germ to homogeneity. The native M r of the enzyme was estimated by gel filtration to be 75 000. The enzyme seems to be a homodimer with a subunit M r of 36 000. No evidence was obtained for the existence of isoforms of the enzyme. The apparent K m values were determined to be 0.24 mM for homoserine and 0.33 mM for ATP. The enzyme activity was not significantly influenced by any of the aspartate-derived amino acids (threonine, methionine, isoleucine, lysine) at least at physiologically relevant concentrations, nor by l- S-adenosylmethionine which is known to be a regulatory metabolise of the pathway.

Evolutionary comparisons of three enzymes of the threonine biosynthetic pathway among several microbial species

As an approach in the study of the evolution of threonine biosynthetic pathways throughout various organisms, the sequences of three enzymes, namely homoserine dehydrogenase, homoserine kinase and threonine synthase, originating from six organisms, namely Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Brevibacterium lactofermentum, Pseudomonas aeruginosa and Saccharomyces cerevisiae, were compared. As a general trend all three enzymatic activities were carried out by proteins sharing sequence relatedness (except for the homoserine kinase of P aeruginosa). Unexpectedly however, for each step one or two enzymes stood out of the main stream: i) for homoserine dehydrogenase, the yeast protein is atypically similar to the E coli enzyme; ii) for homoserine kinase, the P aeruginosa protein shares no similarity with any other species; and iii) for threonine synthase, the B subtilis protein is far distant from the enzymes of other species. Hence in contrast to other biosynthetic pathways such as the tryptophan one, the threonine pathway seems not to have evolved as a whole throughout different organisms but rather each step seems to have been subjected to multiple constraints including substrate-mediated ones and host-specific ones.

Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.

Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes.

Three genes from Pseudomonas aeruginosa involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrc genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The thrB gene was located by pulsed-field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species.

Expression of Escherichia coli homoserine kinase in mouse 3T3 cells.

The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

Isolation of auxotrophic mutants from acetobacter methanolicus MB 58

AbstractThe 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.

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

The hom-thrB operon (homoserine dehydrogenase/homoserine kinase) and the thrC gene (threonine synthase) of Corynebacterium glutamicum ATCC 13,032 and the homFBR (homoserine dehydrogenase resistant to feedback inhibition by threonine) alone as well as homFBR-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 homFBR-thrB as well as of homFBR 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 homFBR and thrB had no effect on threonine or lysine formation in all recombinant strains tested.