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     

Engineering the homoserine dehydrogenase and threonine dehydratase control points to analyse flux towards L-isoleucine in Corynebacterium glutamicum

 The synthesis of L-isoleucine by Corynebacterium glutamicum involves 11 reaction steps, with at least 5 of them regulated in activity or expression. Using gene replacement we constructed a vector-free C. glutamicum strain having feedback-resistant aspartate kinase and feedback-resistant homoserine dehydrogenase activity. Isogenic strains carrying in addition one or several copies of feedback-resistant threonine dehydratase were made and their product accumulations compared. With strain SM1, with high threonine dehydratase activity, accumulation of 50 mM L-isoleucine was achieved, whereas with the parent strain only 4 mM L-isoleucine was obtained. Applying a closed-loop control fed-batch strategy to strain SM1 a final titre of 138 mM L-isoleucine was achieved with an integral molar yield of 0.11 mol/mol, and a maximal specific productivity of 0.28 mmol (g h)^-1. This shows that high L-isoleucine yields can be obtained in the presence of one copy of feedback-resistant homoserine dehydrogenase by applying the appropriate fermentation strategy. In addition, the specific profiles of 2-oxoglutarate and pyruvate accumulation during fermentation revealed a major transition of the metabolism of C. glutamicum during the fermentation process. Received: 16 October 1995/Received revision: 21 December 1995/Accepted: 8 January 1996     

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.     

Expression of the threonine operon fromEscherichia coli inBrevibacterium flavum andCorynebacterium glutamicum

Summary The threonine operon fromEscherichia coli was cloned in plasmid pBR322, subcloned into the shuttle vector pCEM300 and the resulting recombinant plasmid was transferred intoBrevibacterium flavum andCorynebacterium glutamicum. The expression ofE. coli threonine genes in these coryneform bacteria was demonstrated by complementing thethrA andthrB mutations and by assaying homoserine dehydrogenase activity.     

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     

Use of Feedback-Resistant Threonine Dehydratases of Corynebacterium glutamicum To Increase Carbon Flux towards l-Isoleucine

The biosynthesis of l-isoleucine proceeds via a highly regulated reaction sequence connected with l-lysine and l-threonine synthesis. Using defined genetic Corynebacterium glutamicum strains characterized by different fluxes through the homoserine dehydrogenase reaction, we analyzed the influence of four different ilvA alleles (encoding threonine dehydratase) in vectors with two different copy numbers on the total flux towards l-isoleucine. For this purpose, 18 different strains were constructed and analyzed. The result was that unlike ilvA in vectors with low copy numbers, ilvA in high-copy-number vectors increased the final l-isoleucine yield by about 20%. An additional 40% increase in l-isoleucine yield was obtained by the use of ilvA alleles encoding feedback-resistant threonine dehydratases. The strain with the highest yield was characterized by three hom(Fbr) copies encoding feedback-resistant homoserine dehydrogenase and ilvA(Fbr) encoding feedback-resistant threonine dehydratase on a multicopy plasmid. It accumulated 96 mM l-isoleucine, without any l-threonine as a by-product. The highest specific productivity was 0.052 g of l-isoleucine per g of biomass per h. This comparative flux analysis of isogenic strains showed that high levels of l-isoleucine formation from glucose can be achieved by the appropriate balance of homoserine dehydrogenase and threonine dehydratase activities in a strain background with feedback-resistant aspartate kinase. However, still-unknown limitations are present within the entire reaction sequence.     

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     

Threonine-sensitive aspartate kinase and homoserine dehydrogenase from Pisum sativum

Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg²⁺ or Mn²⁺, and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and K_i for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine.     

Improved L-lysine yield with Corynebacterium glutamicum: use of dapA resulting in increased flux combined with growth limitation

The amino acid L-lysine is produced on a large scale using mutants of Corynebacterium glutamicum. However, as yet recombinant DNA techniques have not succeed in improving strains selected for decades by classic mutagenesis for high productivity. We here report that seven biosynthetic enzymes were assayed and oversynthesis of the dihydrodipicolinate synthase resulted in an increase of lysine accumulation from 220 mM to 270 mM. The synthase, encoded by dapA, is located at the branch point of metabolite distribution to either lysine or threonine and competes with homoserine dehydrogenase for the common substrate aspartate semialdehyde. When graded dapA expression was used, as well as quantification of enzyme activities, intracellular metabolite concentrations and flux rates, a global response of the carbon metabolism to the synthase activity became apparent: the increased flux towards lysine was accompanied by a decreased flux towards threonine. This resulted in a decreased growth rate, but increased intracellular levels of pyruvate-derived valine and alanine. Therefore, modulating the flux at the branch point results in an intrinsically introduced growth limitation with increased intracellular precursor supply for lysine synthesis. This does not only achieve an increase in lysine yield but this example of an intracellularly introduced growth limitation is proposed as a new general means of increasing flux for industrial metabolite overproduction. Received: 8 August 1997 / Received revision: 2 October 1997 / Accepted: 14 October 1997     

Expression of the Escherichia coli Catabolic Threonine Dehydratase in Corynebacterium glutamicum and Its Effect on Isoleucine Production

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1.16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to α-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.     

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.     

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.     

Lysine overproducing Corynebacterium glutamicum is characterized by a robust linear combination of two optimal phenotypic states

A homoserine auxotroph strain of Corynebacterium glutamicum accumulates storage compound trehalose with lysine when limited by growth. Industrially lysine is produced from C. glutamicum through aspartate biosynthetic pathway, where enzymatic activity of aspartate kinase is allosterically controlled by the concerted feedback inhibition of threonine plus lysine. Ample threonine in the medium supports growth and inhibits lysine production (phenotype-I) and its complete absence leads to inhibition of growth in addition to accumulating lysine and trehalose (phenotype-II). In this work, we demonstrate that as threonine concentration becomes limiting, metabolic state of the cell shifts from maximizing growth (phenotype-I) to maximizing trehalose phenotype (phenotype-II) in a highly sensitive manner (with a Hill coefficient of 4). Trehalose formation was linked to lysine production through stoichiometry of the network. The study demonstrated that the net flux of the population was a linear combination of the two optimal phenotypic states, requiring only two experimental measurements to evaluate the flux distribution. The property of linear combination of two extreme phenotypes was robust for various medium conditions including varying batch time, initial glucose concentrations and medium osmolality.     

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.     

Threonine Locus of Escherichia coli K-12: Genetic Structure and Evidence for an Operon

Three genes, thrA, thrB, and thrC, were previously defined and localized in the threonine locus of Escherichia coli K-12. thrA, thrB, and thrC specify the enzymes aspartokinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase, respectively. A complementation analysis of the threonine cluster using derivatives of a lambda phage carrying the threonine genes (lambdadthr(c)) demonstrates that: (i) thrB and thrC each consist of a single cistron; and (ii) thrA is composed of two cistrons, thrA(1) and thrA(2), although it specifies a single polypeptide chain. thrA(1) and thrA(2) correspond to aspartokinase I and homoserine dehydrogenase I, respectively. Their relative order is established. The demonstration of polar effects of mutations (nonsense or induced by phage Mu) in thrA and thrB is taken as evidence for the existence of a thrA thrB thrC operon, transcribed in this order.     

Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum

The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.