Flux through the tetrahydrodipicolinate succinylase pathway is dispensable for l-lysine production in Corynebacterium glutamicum

The N-succinyl-ll-diaminopimelate desuccinylase gene (dapE) in the four-step succinylase branch of the l-lysine biosynthetic pathway of Corynebacterium glutamicum was disrupted via marker-exchange mutagenesis to create a mutant strain that uses only the one-step meso-diaminopimelate dehydrogenase branch to overproduce lysine. This mutant strain grew and utilized glucose from minimal medium at the same rate as the parental strain. In addition, the dapE  ⁻ strain produced lysine at the same rate as its parent strain. Transformation of the parental and dapE  ⁻ strains with the amplified meso-diaminopimelate dehydrogenase gene (ddh) on a plasmid did not affect lysine production in either strain, despite an eightfold amplification of the activity of the enzyme. These results indicate that the four-step succinylase pathway is dispensable for lysine overproduction in shake-flask culture. In addition, the one-step meso-diaminopimelate dehydrogenase pathway does not limit lysine flux in Corynebacterium under these conditions. Received: 20 May 1998 / Received revision: 12 August 1998 / Accepted: 3 September 1998     

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     

Overexpression of cytosolic malate dehydrogenase (MDH2) causes overproduction of specific organic acids in Saccharomyces cerevisiae

Saccharomyces cerevisiae accumulates l-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a 6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in l-malic acid accumulation in the production medium. The high apparent K _m of MDH2 for l-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function of the enzyme and differs from the previously published K _m of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be a limiting factor, thus providing a system for further metabolic engineering of l-malic acid production. The overexpression of MDH2 activity also causes an elevation in the accumulation of fumaric acid and citric acid. Accumulation of fumaric acid is presumably caused by high intracellular l-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing possibility that cytosolic l-malic acid is a direct precursor of citric acid in yeast. Received: 22 January 1997 / Received revision: 14 April 1997 / Accepted: 19 April 1997     

C3-Carboxylation as an anaplerotic reaction in phosphoenolpyruvate carboxylase-deficient Corynebacterium glutamicum

Phosphoenolpyruvate carboxylase (PEPCx) has recently been found to be dispensable as an anaplerotic enzyme for growth and lysine production of Corynebacterium glutamicum. To clarify the role of the glyoxylate cycle as a possible alternative anaplerotic sequence, defined PEPCx- and isocitrate-lyase (ICL)-negative double mutants of C. glutamicum wild-type and of the l-lysine-producing strain MH20-22B were constructed by disruption of the respective genes. Analysis of these mutants revealed that the growth on glucose and the lysine productivity were identical to that of the parental strains. These results show that PEPCx and the glyoxylate cycle are not essential for growth of C. glutamicum on glucose and for lysine production and prove the presence of another anaplerotic reaction in this organism. To study the anaplerotic pathways in C. glutamicum further, H¹³CO₃ ^–-labeling experiments were performed with cells of the wild-type and a PEPCx-negative strain growing on glucose. Proton nuclear magnetic resonance analysis of threonine isolated from cell protein of both strains revealed the same labeling pattern: about 37% ¹³C enrichment in C-4 and 3.5% ¹³C enrichment in C-1. Since the carbon backbone of threonine corresponds to that of oxaloacetate, the label in C-4 of threonine positively identifies the anaplerotic pathway as a C₃-carboxylation reaction that also takes place in the absence of PEPCx. Received: 27 December 1995 / Accepted: 20 March 1996     

Improved L-lysine production by the amplification of theCorynebacterium glutamicum dapA gene encoding dihydrodipicolinate synthetase inE. coli

Summary ThedapA gene (L-2,3-dihydrodipicolinate synthetase: DHDP synthetase) ofCorynebacterium glutamicum JS231, a lysine overproducer, cloned and subcloned inE. coli/C. glutamicum shuttle vector pECCG117 was used to transformE. coli threonine producer and threonine and lysine coproducer. The plasmid pDHDP5812 carryingdapA gene ofC. glutamicum led to increase in lysine production in theseE. coli strains. Threonine and lysine co-producerE. coli TF1 with pDHDP5812 produced lysine with small amount of threonine. The DHDP synthetase activity ofE. coli TF1 carrying pDHDP5812 showed high resistance toward inhibition by lysine.     

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     

Poly-3-hydroxybutyrate production by washed cells of Alcaligenes eutrophus; purification, characterisation and potential regulatory role of citrate synthase

Washed cells prepared from carbon-limited continuous cultures of Alcaligenes eutrophus synthesised poly-3-hydroxybutyrate (PHB) rapidly when supplied with glucose, dl-lactate or l-lactate. Unlike growing cultures, washed cells excreted significant amounts of pyruvate. The combined rates of PHB production (qPHB) and pyruvate excretion (qPyr) were linearly related to the rate of carbon substrate utilisation (qS), showing that washed cells behaved similarly to growing cultures when corrected for the absence of non-PHB biomass production. The addition of formate (as a potential source of NADH and/or ATP) significantly stimulated both qPHB and qPyr, but slightly decreased qS and substantially decreased the flux of carbon through the tricarboxylic acid cycle (qTCA). Citrate synthase activity of broken cells was inhibited by physiological concentrations of NADH, but not of ATP, in a manner that was not reversible by AMP. Citrate synthase was purified and shown to be a “large” form of the enzyme (M _r 227,000), comprising a single type of subunit (M _r 47,000) as found in several other gram-negative aerobes. The potential role of citrate synthase in the regulation of PHB production via its ability to control carbon flux into the tricarboxylic acid cycle is discussed. Received: 14 March 1997 / Accepted: 9 July 1997     

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.     

Biotransformation of 1-naphthalenesulfonic acid by the green alga Scenedesmus obliquus

Under sulfate limitation, axenic batch cultures of the green alga Scenedesmus obliquus metabolized 1-naphthalenesulfonic acid and partially used the sulfonate as a source of sulfur. The main metabolite, 1-hydroxy-2-naphthalenesulfonic acid, which was not metabolized further in the algal culture, was formed by hydroxylation of the substrate in position 1 and by migration of the sulfonic acid group to position 2 of the naphthalene ring (NIH shift). A smaller amount of 1-naphthalenesulfonic acid was desulfonated. The resulting 1-naphthol was mostly transformed into 1-naphthyl β-d-glucopyranoside. Received: 27 March 1996 / Revision received: 18 October 1996 / Accepted: 30 October 1996     

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation. Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997     

Efficient shoot organogenesis of eggplant (Solanum melongena L.) induced by thidiazuron

Morphogenesis from several explant types excised from in-vitro-grown plantlets of a Brazilian eggplant (Solanum melongena L.) variety (F-100) was evaluated in response to thidiazuron (TDZ). Leaves and cotyledons were found to be the most responsive explants. Optimal shoot bud induction rates (75–100 buds/explant) were achieved in the presence of 0.2 μm TDZ. Organogenic calli were transferred to growth regulator free MS medium before shoot excision. Rooting was induced on half-strength MS medium supplemented with 0.6 μm IAA. Received: 1 March 1997 / Revision received: 29 October 1997 / Accepted: 15 November 1997     

Novel l-specific cleavage of the urethane bond of t -butoxycarbonylamino acids by whole cells of Corynebacterium aquaticum

Microorganisms capable of cleaving the urethane bond of t-butoxycarbonyl (Boc) amino acids in a whole-cell reaction were screened among stock cultures, and Corynebacterium aquaticum IFO12154 was the most promising. The conversion of Boc-Ala to Ala was stimulated by CoSO₄ in the medium and reaction mixture. The optimum whole-cell concentration was 25 mg lyophilized cells/ml. Boc-l-Met was the best substrate for this reaction, and other Boc-L-amino acids, as well as benzyloxycarbonyl-l-amino acids with hydrophobic residues, were also good substrates. Boc-d- and Z-d-amino acids were inert. When the reactions had proceeded for 24 h with each substrate at 10 mM, the molar conversion rates from Boc-l-, dl- and d-Met were 100%, 50%, and 0% respectively. From 150 mM Boc-l-Met, 143 mM l-Met was formed at a molar yield of 95.3%. Received: 3 September 1996 / Received last revision: 7 April 1997 / Accepted: 19 April 1997     

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     

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165–170 nmol min^–1 (mg protein)^–1] were found in cells grown on acetate or β-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50–80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent K _m of isocitrate lyase for d,l-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 μM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the V _max nor the d,l-isocitrate K _m of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum. Received: 10 October 1997 / Accepted: 16 January 1998     

A method to increase silver biosorption by an industrial strain of Saccharomyces cerevisiae

 Ag⁺ biosorption by an industrial strain of Saccharomyces cerevisiae was investigated. Older (96 h old) biomass had half the biosorption capacity of younger (24 h old) biomass (0.187 and 0.387 mmol Ag⁺/g dry mass respectively). Comparisons of cell walls isolated from biomass of either age indicated that chemical composition and Ag⁺ biosorption capacity varied little over the time span examined and that cell walls from either age of culture had small Ag⁺ biosorption capacities compared to whole cells of a similar age. Silver-containing precipitates were observed both on the cell wall and within the cell, indicating that intracellular components sorbed Ag⁺. The concentration of these precipitates within the cell appeared visually to decrease with age in Ag⁺-exposed cells. Incorporation of L-cysteine into the growth medium resulted in biomass with increased silver biosorption capacities, protein and sulphydryl group content. Increasing the concentration of L-cysteine in the growth medium from 0 to 5.0 mM increased silver biosorption from 0.389 to 0.556 mmol Ag⁺/g dry mass Isolated cell walls of biomass grown in supplemented media also showed a possible link between silver biosorption capacities, protein and sulphydryl group content. No precipitates were observed in silver-exposed biomass that had been grown in the presence of 5.0 mM L-cysteine. Received: 12 April 1995 / Received revision: 7 August 1995 / Accepted: 22 August 1995     

Effects of <Emphasis Type="Italic">dapA</Emphasis> gene deletion on coronatine biosynthesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">glycinea</Emphasis> PG4180

Coronatine (COR) is a structural and functional analogue of jasmonic acid that might be employed in agriculture to elicit plant resistance against various aggressors. However, the yield of COR is low both in chemosynthesis and biosynthesis, so broad investigation of COR is difficult. Coronatine combines two distinct components: coronafacic acid (CFA) and coronamic acid (CMA). Synthesis of both CMA and CFA is involved in l-isoleucine metabolism, so the objective of this work was to investigate if COR production can be improved by regulating amino acid biosynthesis in P. syringae pv. glycinea. Inhibition of dihydrodipicolinate synthase was achieved by removing the dapA gene via homologous recombination, which resulted in a COR yield by the dapA ⁻ mutant of about 1.5-fold greater than the wild strain. Thus, regulation of amino acid metabolism is a feasible way to increase COR production, which could be a more effective method than adding substrates into culture medium.