Influence of threonine exporters on threonine production in Escherichia coli

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.     

Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-defective mutants.

We found that the simple addition of L-methionine to the wild type of Corynebacterium glutamicum results in excretion of the cellular building block L-lysine up to rates of 2.5 nmol/min/mg (dry weight). Biochemical analyses revealed that L-methionine represses the homoserine dehydrogenase activity and reduces the intracellular L-threonine level from 7 to less than 2 mM. Since L-lysine synthesis is regulated mainly by L-threonine (plus L-lysine) availability, the result is enhanced flux towards L-lysine. This indicates a delicate and not well controlled type of flux control at the branch point of aspartate semialdehyde conversion to either L-lysine or L-threonine, probably due to the absence of isoenzymes in C. glutamicum. The inducible system of L-lysine excretion discovered was used to isolate mutants defective in the excretion of this amino acid. One such mutant characterized in detail accumulated 174 mM L-lysine in its cytosol without extracellular excretion of L-lysine, whereas the wild type accumulated 53 mM L-lysine in the cytosol and 5.9 mM L-lysine in the medium. The mutant was unaffected in L-lysine uptake or L-isoleucine or L-glutamate excretion, and also the membrane potential was unaltered. This mutant therefore represents a strain with a defect in an excretion system for the primary metabolite L-lysine.     

Identification of glyA (encoding serine hydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum

L-threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum. However, in the course of this process, some of the L-threonine is degraded to glycine. We detected an aldole cleavage activity of L-threonine in crude extracts with an activity of 2.2 nmol min(-1) (mg of protein)(-1). In order to discover the molecular reason for this activity, we cloned glyA, encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged glyA, SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with L-threonine as the substrate was 1.3 micromol min(-1) (mg of protein)(-1), which was 4% of that with L-serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P(tac), making glyA expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an L-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while L-threonine was simultaneously increased by 49%. The intracellular availability of L-threonine to aldole cleavage was also reduced by overexpressing the L-threonine exporter thrE. In C. glutamicum DR-17, which overexpresses thrE, accumulation of 67 mM instead of 49 mM L-threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.     

Effect of transport proteins on l-isoleucine production with the l-isoleucine-producing strain Corynebacterium glutamicum YILW

Previous studies have shown that the deletion of brnQ from the Corynebacterium glutamicum chromosome results in a significant reduction in L-isoleucine uptake rates, while overexpression of brnFE leads to enhanced L-isoleucine export rates. Given that net excretion rates would be an important factor for high titers of L-isoleucine accumulation, we have tested the notion that decreased L-isoleucine uptake combined with increased L-isoleucine excretion will further improve high-yield strains that are currently used for the industrial-scale production of L-isoleucine. To examine the effect of the two carriers on L-isoleucine accumulation in L-isoleucine producer C. glutamicum YILW, we constructed a brnQ deletion mutant (C. glutamicum YILW?brnQ) and two brnFE overexpressors (C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW?brnQpXMJ19brnFE). Compared to the original strain, the efflux rate of the brnQ mutant increased from 19.0 to 23.6?nmol?min(-1) mg (dry wt)(-1) and its L-isoleucine titer increased from 154.3?mM (20.2?g?l(-1)) to 170.3?mM (22.3?g?l(-1)). The efflux rates of C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW?brnQpXMJ19brnFE were 33.5 and 39.1?nmol?min(-1) mg (dry wt)(-1), and their L-isoleucine production titers were 197.2?mM (25.9?g?l(-1)) and 221.0?mM (29.0?g?l(-1)), respectively. Our results suggest that modifications of the transport system could provide a promising avenue for further increasing L-isoleucine yield in the L-isoleucine producer.     

L-threonine export: use of peptides to identify a new translocator from Corynebacterium glutamicum

Bacterial mechanisms for the uptake of peptides and their hydrolysis to amino acids are known in great detail, whereas much less is known about the fates of the peptide-derived amino acids. We show that the addition of L-threonine-containing di- or tripeptides results in reduction of the growth of Corynebacterium glutamicum, with concomitant high intracellular accumulation of L-threonine to up to 130 mM. Using transposon mutagenesis and isolation of mutants with increased Thr peptide sensitivity, nine open reading frames (ORFs) were identified, almost all encoding hypothetical proteins of unknown function. Three ORFs encode membrane proteins. Their individual functional characterizations in the wild-type background led to the identification of thrE. Upon thrE overexpression, growth is no longer sensitive to the presence of the Thr peptide, and L-threonine is exported at a rate of 3.8 nmol min(-1) mg of dry weight(-1), whereas the rate of export of a thrE inactivation mutant is reduced to 1.1 nmol min(-1) mg of dry weight(-1). In addition to L-threonine, L-serine is also a substrate for the exporter. The exporter exhibits nine predicted transmembrane-spanning helices with long charged C and N termini and with an amphipathic helix present within the N terminus. All these data suggest that the carrier encoded by thrE serves to export small molecules such as L-threonine and that the carrier is a prototype of a new translocator family. Homologues of ThrE are present in Mycobacterium tuberculosis and Streptomyces coelicolor.     

Quantitative discrimination of carrier-mediated excretion of isoleucine from uptake and diffusion in Corynebacterium glutamicum.

The efflux of isoleucine in whole cells of Corynebacterium glutamicum was studied. The different amino acid fluxes across the plasma membrane were functionally discriminated into passive diffusion, carrier-mediated excretion, and carrier-mediated uptake. Detailed kinetic analysis was made possible by controlled variation of internal isoleucine from low concentrations to 100 mM by feeding with mixtures of isoleucine-containing peptides. Isoleucine diffusion was experimentally separated and proceeded with a first-order rate constant of 0.083 min-1 or 0.13 microliters.min-1.mg (dry mass)-1, which corresponds to a permeability of 2 x 10(-8) cm.s-1. Uptake of isoleucine was constant at a rate of 1.1 nmol.min-1.mg (dry mass)-1. Carrier-mediated isoleucine excretion was zero below a threshold of 8 mM cytosolic isoleucine. Above this level, a Michaelis-Menten-type kinetics was observed, with a Km of 21 mM (13 mM plus 8 mM threshold value) and a Vmax of 14.5 nmol.min-1.mg (dry mass)-1. The activity of the isoleucine excretion carrier depended on the presence of a membrane potential. Excretion was specific for L-isoleucine (and presumably L-leucine) and could be inhibited by SH reagents.     

A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by L-threonine.

In Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum, homoserine dehydrogenase (HD), the enzyme after the branch point of the threonine/methionine and lysine biosynthetic pathways, is allosterically inhibited by L-threonine. To investigate the regulation of the C. glutamicum HD enzyme by L-threonine, the structural gene, hom, was mutated by UV irradiation of whole cells to obtain a deregulated allele, homdr. L-Threonine inhibits the wild-type (wt) enzyme with a Ki of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM L-threonine. The homdr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned homdr gene is genetically unstable. The cloned homdr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM L-threonine. Sequence analysis revealed that the homdr mutation is a single nucleotide (G1964) deletion in codon 429 within the hom reading frame. The resulting frame-shift mutation radically alters the structure of the C terminus, resulting in ten amino acid (aa) changes and a deletion of the last 7 aa relative to the wt protein. These observations suggest that the C terminus may be associated with the L-threonine allosteric response. The homdr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.     

L-valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum

Corynebacterium glutamicum was engineered for the production of L-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum DeltaaceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, L-alanine, and L-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum DeltaaceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and L-alanine towards L-valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum DeltaaceE(pJC4ilvBNCE) produced up to 210 mM L-valine with a volumetric productivity of 10.0 mM h(-1) (1.17 g l(-1) h(-1)) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose.     

Investigations into enzymes of nitrogen metabolism of the ectomycorrhizal basidiomycete, Suillus bovinus

Axenic mycelia of the ectomycorrhizal basidiomycete, Suillus bovinus, were grown in liquid media under continuous aeration with compressed air at 25 degrees C in darkness. Provided with glucose as the only carbohydrate source, they produced similar amounts of dry weight with ammonia, with nitrate or with alanine, 60-80% more with glutamate or glutamine, but about 35% less with urea as the respectively only exogenous nitrogen source. In crude extracts of cells from NH4(+)-cultures, NADH-dependent glutamate dehydrogenase exhibited high aminating (688 nmol x mg protein(-1) x min(-1)) and low deaminating (21 nmol x mg protein(-1) x min(-1)) activities. Its Km-values for 2-oxoglutarate and for glutamate were 1.43 mM and 23.99 mM, respectively. pH-optimum for amination was about 7.2, that for deamination about 9.3. Glutamine synthetase activity was comparatively low (59 nmol x mg protein(-1) x min(-1)). Its affinity for glutamate was poor (Km = 23.7 mM), while that for the NH4+ replacing NH2OH was high (Km = 0.19 mM). pH-optimum was found at 7.0. Glutamate synthase (= GOGAT) revealed similar low activity (62 nmol x mg protein(-1) x min(-1)), Km-values for glutamine and for 2-oxoglutarate of 2.82 mM and 0.28 mM, respectively, and pH-optimum around 8.0. Aspartate transaminase (= GOT) exhibited similar affinities for aspartate (Km = 2.55 mM) and for glutamate (Km = 3.13 mM), but clearly different Km-values for 2-oxoglutarate (1.46 mM) and for oxaloacetate (0.13 mM). Activity at optimum pH of about 8.0 was 506 nmol x mg protein(-1) x min(-1) for aspartate conversion, but only 39 nmol x mg protein(-1) x min(-1) at optimum pH of about 7.0 for glutamate conversion. Activity (599 nmol x mg protein(-1) x min(-1)), substrate affinities (Km for alanine = 6.30 mM, for 2-oxoglutarate = 0.45 mM) and pH-optimum (6.5-7.5) proved alanine transaminase (= GPT) also important in distribution of intracellular nitrogen. There was comparatively low activity of the obviously constitutive enzyme, urease, (42 nmol x mg protein(-1) x min(-1)) whose substrate affinity was rather high (Km = 0.56 mM). Nitrate reductase proved substrate induced; activity could only be measured after exposure of the mycelia to exogenous nitrate. Routes of entry of exogenous nitrogen and tentative significance of the various enzymes in cell metabolism are discussed.     

Cometabolism of a nongrowth substrate: L-serine utilization by Corynebacterium glutamicum

Despite its key position in central metabolism, L-serine does not support the growth of Corynebacterium glutamicum. Nevertheless, during growth on glucose, L-serine is consumed at rates up to 19.4 +/- 4.0 nmol min(-1) (mg [dry weight])(-1), resulting in the complete consumption of 100 mM L-serine in the presence of 100 mM glucose and an increased growth yield of about 20%. Use of 13C-labeled L-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton of L-serine is mainly converted to pyruvate-derived metabolites such as L-alanine. The sdaA gene was identified in the genome of C. glutamicum, and overexpression of sdaA resulted in (i) functional L-serine dehydratase (L-SerDH) activity, and therefore conversion of L-serine to pyruvate, and (ii) growth of the recombinant strain on L-serine as the single substrate. In contrast, deletion of sdaA decreased the L-serine cometabolism rate with glucose by 47% but still resulted in degradation of L-serine to pyruvate. Cystathionine beta-lyase was additionally found to convert L-serine to pyruvate, and the respective metC gene was induced 2.4-fold under high internal L-serine concentrations. Upon sdaA overexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellular L-serine conversion to pyruvate might occur, although probably the weak affinity of L-SerDH (apparent Km, 11 mM) prevents substantial L-serine degradation.     

Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate.

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose

Growth of Corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By (13)C-labeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on acetate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol. (mg of protein)(-1). min(-1), respectively. The citric acid cycle was replenished by carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate (30 nmol. [mg of protein](-1). min(-1)) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on glucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol. [mg of protein](-1). min(-1)). Surprisingly, the anaplerotic function was fulfilled completely by the glyoxylate cycle (50 nmol. [mg of protein](-1). min(-1)) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of C. glutamicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.     

Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum

A corynebacterial clone, previously isolated by scoring repression of lacZYA fused to the aceB promoter of Corynebacterium glutamicum, was analyzed further. In the clone, an open reading frame designated glxR, consisting of 681 nucleotides and encoding a 24,957-Da protein, was found. The molecular mass of a native GlxR protein was estimated by gel filtration column chromatography to be 44,000 Da, suggesting that the protein formed dimers. The predicted amino acid sequence contained both cyclic AMP (cAMP)- and DNA-binding motifs and was homologous with the cAMP receptor protein family of proteins. The aceB-repressing activity of the glxR clone was markedly relieved in an Escherichia coli cya mutant, but the activity was restored in growth medium containing cAMP. In glucose medium, the intracellular cAMP concentration of C. glutamicum reached 22 nmol/mg of protein in the early exponential phase and then decreased further; but in acetate medium, the intracellular cAMP concentration was only 5 nmol/mg of protein and remained low throughout the growth phase. The expression of glxR was not affected by the carbon source. Binding of purified GlxR to the promoter region of aceB could be demonstrated only in the presence of cAMP. These data suggest that GlxR may form dimers which bind to the aceB promoter region in the presence of cAMP and repress the glyoxylate bypass genes.     

The PduX enzyme of Salmonella enterica is an L-threonine kinase used for coenzyme B12 synthesis

Here, the PduX enzyme of Salmonella enterica is shown to be an L-threonine kinase used for the de novo synthesis of coenzyme B(12) and the assimilation of cobyric acid (Cby). PduX with a C-terminal His tag (PduX-His(6)) was produced at high levels in Escherichia coli, purified by nickel affinity chromatography, and partially characterized. (31)P NMR spectroscopy established that purified PduX-His(6) catalyzed the conversion of l-threonine and ATP to L-threonine-O-3-phosphate and ADP. Enzyme assays showed that ATP was the preferred substrate compared with GTP, CTP, or UTP. PduX displayed Michaelis-Menten kinetics with respect to both ATP and l-threonine and nonlinear regression was used to determine the following kinetic constants: V(max) = 62.1 +/- 3.6 nmol min(-1) mg of protein(-1); K(m)(, ATP) = 54.7 +/- 5.7 microm and K(m)(,Thr) = 146.1 +/- 8.4 microm. Growth studies showed that pduX mutants were impaired for the synthesis of coenzyme B(12) de novo and from Cby, but not from cobinamide, which was the expected phenotype for an L-threonine kinase mutant. The defect in Cby assimilation was corrected by ectopic expression of pduX or by supplementation of growth medium with L-threonine-O-3-phosphate, providing further support that PduX is an L-threonine kinase. In addition, a bioassay showed that a pduX mutant was impaired for the de novo synthesis of coenzyme B(12) as expected. Collectively, the genetic and biochemical studies presented here show that PduX is an L-threonine kinase used for AdoCbl synthesis. To our knowledge, PduX is the first enzyme shown to phosphorylate free L-threonine and the first L-threonine kinase shown to function in coenzyme B(12) synthesis.     

Identification and characterization of γ-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabP(Cg)) encoded by ncgl0464 was identified and characterized. GabP(Cg) plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabP(Cg) was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na(+)). The K(m) and V(max) values were determined to be 41.1 ± 4.5 μM and 36.8 ± 2.6 nmol min(-1) (mg dry weight [DW])(-1), respectively, at pH 6.5 and 34.2 ± 1.1 μM and 67.3 ± 1.0 nmol min(-1) (mg DW)(-1), respectively, at pH 7.5. GabP(Cg) has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli GabP and Bacillus subtilis GabP, respectively). The mutant RES167 Δncgl0464/pGXKZ9 with the GabP(Cg) deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabP(Cg) represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains.     

Contraction of human airways by oxidative stress protection by N-acetylcysteine.

We examined the in vitro effects of tert-butylhydroperoxide (tBu-OOH) in human bronchial muscle. tert-Butylhydroperoxide produced concentration-dependent contractions of bronchial rings (maximum effect was 56.5 +/- 9.6% of contraction by 1 mM acetylcholine; effective concentration 50% was approximately 100 microM). tert-Butylhydroperoxide (0.5 mM)-induced contraction was enhanced by epithelial removal but abolished by indomethacin (cyclooxygenase inhibitor) and zileuton (lipoxygenase inhibitor). tert-Butylhydroperoxide produced a transient rise in intracellular calcium in human cultured airway smooth muscle cells (HCASMC). The bronchial reactivity to acetylcholine and histamine was not altered by tBu-OOH. In HCASMC, tBu-OOH (0.5 mM, 30 min) increased malondialdehyde levels (MDA; from 7.80 +/- 0.83 to 26.82 +/- 1.49 nmol mg(-1) protein), accompanied by a decrease of reduced glutathione (GSH; from 16.7 +/- 2.6 to 6.9 +/- 1.9 nmol mg(-1) protein) and an increase of oxidized glutathione (from 0.09 +/- 0.03 to 0.18 +/- 0.03 nmol mg(-1) protein). N-acetylcysteine (0.3 mM) inhibited by approximately 60% the bronchial contraction resulting from tBu-OOH (0.5 mM) and protected cultured cells exposed to tBu-OOH (MDA was lowered to 19.51 +/- 1.19 nmol mg(-1) protein, and GSH content was replenished). In summary, tBu-OOH caused contraction of human bronchial muscle mediated by release of cyclo-oxygenase and lipoxygenase products without producing airways hyperreactivity. N-acetylcysteine decreases tBu-OOH-induced contraction and protects human cultured airway smooth muscle cells exposed to tBu-OOH.     

Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine.

An approach to broaden the product range of the ethanologenic, gram-negative bacterium Zymomonas mobilis by means of genetic engineering is presented. Gene alaD for L-alanine dehydrogenase (EC 1.4.1.1.) from Bacillus sphaericus was cloned and introduced into Z. mobilis. Under the control of the strong promoter of the pyruvate decarboxylase (pdc) gene, the enzyme was expressed up to a specific activity of nearly 1 mu mol . min -1 . mg of protein -1 in recombinant cells. As a results of this high L-alanine dehydrogenase activity, growing cells excreted up to 10 mmol of alanine per 280 mmol of glucose utilized into a mineral salts medium. By the addition of 85 mM NH4+ to the medium, growth of the recombinant cells stopped, and up to 41 mmol alanine was secreted. As alanine dehydrogenase competed with pyruvate decarboxylase (PDC) (EC 4.1.1.1.) for the same substrate (pyruvate), PDC activity was reduced by starvation for the essential PDC cofactor thiamine PPi. A thiamine auxotrophy mutant of Z. mobilis which carried the alaD gene was starved for 40 h in glucose-supplemented mineral salts medium and then shifted to mineral salts medium with 85 mM NH4+ and 280 mmol of glucose. The recombinants excreted up to 84 mmol of alanine (7.5 g/liter) over 25 h. Alanine excretion proceeded at an initial velocity of 238 nmol . min-1 . mg [dry weight]-1. Despite this high activity, the excretion rate seemed to be a limiting factor, as the intracellular concentration of alanine was as high as 260 mM at the beginning of the excretion phase and decreased to 80 to 90 mM over 24 h.     

Expression of an L-alanine dehydrogenase gene in Zymomonas mobilis and excretion of L-alanine.

An approach to broaden the product range of the ethanologenic, gram-negative bacterium Zymomonas mobilis by means of genetic engineering is presented. Gene alaD for L-alanine dehydrogenase (EC 1.4.1.1.) from Bacillus sphaericus was cloned and introduced into Z. mobilis. Under the control of the strong promoter of the pyruvate decarboxylase (pdc) gene, the enzyme was expressed up to a specific activity of nearly 1 mu mol . min -1 . mg of protein -1 in recombinant cells. As a results of this high L-alanine dehydrogenase activity, growing cells excreted up to 10 mmol of alanine per 280 mmol of glucose utilized into a mineral salts medium. By the addition of 85 mM NH4+ to the medium, growth of the recombinant cells stopped, and up to 41 mmol alanine was secreted. As alanine dehydrogenase competed with pyruvate decarboxylase (PDC) (EC 4.1.1.1.) for the same substrate (pyruvate), PDC activity was reduced by starvation for the essential PDC cofactor thiamine PPi. A thiamine auxotrophy mutant of Z. mobilis which carried the alaD gene was starved for 40 h in glucose-supplemented mineral salts medium and then shifted to mineral salts medium with 85 mM NH4+ and 280 mmol of glucose. The recombinants excreted up to 84 mmol of alanine (7.5 g/liter) over 25 h. Alanine excretion proceeded at an initial velocity of 238 nmol . min-1 . mg [dry weight]-1. Despite this high activity, the excretion rate seemed to be a limiting factor, as the intracellular concentration of alanine was as high as 260 mM at the beginning of the excretion phase and decreased to 80 to 90 mM over 24 h.     

Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae.

Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min(-1) mg of protein(-1) and had an apparent Km for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min(-1) mg of protein(-1). Hypoxanthine PRTase activity was present at 0.85 nmol min(-1) mg of protein(-1) with an apparent Km for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min(-1) mg of protein(-1). PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8-azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity.