Adsorption studies for the separation ofl-tryptophan froml-serine and indole in a bioconversion medium

l-tryptophan was produced froml-serine and indole by immobilized Escherichia coli cells in organic-aqueous systems. Selective adsorption was the method chosen to enable both product separation andl-serine reutilization. Amongst various adsorbents tested activated carbons and neutral polymeric resins (XAD-4 and XAD-7) showed good performance. The neutral resins could selectively concentrate thel-tryptophan from dilute aqueous solutions and adsorbed only 5% of the unconvertedl-serine. High separation factors (l-tryptophan/l-serine and indole/l-tryptophan) were obtained with these adsorbents. Despite a lower capacity, the XAD-7 resin had the advantage of desorbingl-tryptophan with basic or acidic solutions, while organic solvents were required to desorb, at the same concentration levels, this compound from XAD-4.In a packed bed column filled with XAD-4 resin or activated carbon, totall-tryptophan adsorption and recovery were achieved at linear velocities up to 5.0 cm/min and 3.2 cm/min respectively. Successive sorbent reutilization, following continuous sorption and elution steps, was carried out in packed bed columns with the neutral resins and activated carbon.Thel-form of tryptophan, after crystallization, was identified by HPTLC.List of Symbols HPLC High Performance Liquid Chromatography - HPTLC High Performance Thin Layer Chromatography - Trp tryptophan - Ser Serine - A amount of sorbent(g) - c equilibrium solute concentration in the aqueous phase (g/dm³) - c _i initial (before adding the sorbent) liquid phase concentration (g/dm³) - C _T tryptophan concentration in the inlet solution (g/dm³) - C _To tryptophan concentration in the outlet solution (g/dm³) - E _z axial dispersion coefficient (m²/s) - k experimental constant (Eq. 1, 2 and 3) - K ₁ rate constant of adsorption (min^–1) - L column length(m) - n experimental constant (eq. 1, 2 and 3) - q equilibrium solid phase concentration (g solute/g sorbent) - q _max maximum capacity of sorbent (g solute/g sorbent) - t time(s) - v liquid velocity (m/s) - V volume of liquid phase(dm³) - V _e eluted volume(dm³) - V _r volume needed to saturate the column (dm³)     

Hyper-production of l-trytophan via fermentation with crystallization

A stable and fast l-tryptophan producer, AGX1757, was isolated from Escherichia coli W3110 trpAE1 trpR tnaA, which carried pSC101-trpI15·14. Cells of AGX1757 did not lose the composite plasmid during fermentation. Whenever a fed-batch culture of AGX1757 attained an l-tryptophan concentration of about 30 g/l, indole began to appear in the broth. The emergence of indole was caused by inhibition of tryptophan synthase due to accumulated l-tryptophan. Hence, the production rate of l-tryptophan sharply decreased. A higher solubility of l-tryptophan in the supernatant of culture broth (about 32 g/l) than that in the initial medium (about 22 g/l) was attributed to some unknown interaction between l-tryptophan and certain macromolecular material(s) coming from the bacterial cells. An addition of non-ionic detergents into the supernatant was effective for decreasing the solubility of l-tryptophan, hence causing crystallization of l-tryptophan. Pluronic L-61 was supplied from outside to an extent of 0.5% in terms of wt% concentration at around 45 h of fermentation when the l-tryptophan accumulated reached about 25 g/l. This addition actually caused crystallization of l-tryptophan and, as a result, the inhibitory effect of tryptophan synthase by l-tryptophan accumulated in the broth could be alleviated. Thus far, further fermentation became possible. l-Tryptophan of more than 50 g/l was finally produced by feeding solutions of both glucose and anthranilic acid. Correspondence to: H. Tsunekawa     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine production by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach

The l-phenylalanine (l-Phe) production by Escherichia coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem for an improved l-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N’-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in l-Phe production and a remarkable tolerance to 1 × 10¹⁰ pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced l-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH–stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on l-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at μ _set = 0.18 h⁻¹ in the first 20 h and a following linear varying rate feeding with F = (−0.55 × t + 18.6) ml/h, was developed to improve l-Phe production. With this two-stage feeding approach, a maximum l-Phe titer of 57.63 g/l with a high l-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential l-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03).     

Chemoreception in the green algaDunaliella tertiolecta

A capillary assay was employed to quantify positive chemotactic responses in the motile, unicellular, marine algaDunaliella tertiolecta. Among a wide range of inorganic and organic compounds tested, only ammonium ion,l-tyrosine,l-tryptophan, andl-phenylalanine were found to be major atractants for the chlorophyte.l-Methionine andl-cysteine weakly attracted the alga at 10⁻³ M. The minimum concentration of the major attractants needed to elicit an observable chemotactic response was approximately 10⁻⁶ M. The maximum response occurred when the capillaries contained 10⁻⁵ Ml-tyrosine orl-tryptophan, 10⁻⁴ Ml-phenylalanine, and 10⁻³ M ammonium chloride. The other amino acids, carbohydrates, B-vitamins, urea, and nitrate were among the chemicals that failed to attractD. tertiolecta. The alga apparently possesses one chemoreceptor that binds ammonium ion only, and another chemoreceptor that binds the three aromatic amino acids.     

Plant Growth Inhibitory Compounds from Aqueous Leachate of Wheat Straw

When seedlings of lettuce, cress, rice and wheat were incubated with the leachate of wheat straw, the roots growth of lettuce and garden cress were particularly inhibited. The leachate of wheat straw (100 g eq./l) showed 80.5 and 79.4% inhibition for lettuce and cress roots, respectively. The inhibitory activity was stronger as the concentration of wheat straw leachate was greater. This result indicates that allelochemical(s) inhibiting the roots growth of lettuce and cress are leached from the wheat straw into the water. Two potent compounds were isolated from the leachate of the wheat straw and identified as syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan by spectral analyses. Syringoylglycerol 9-O-β-d-glucopyranoside inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 10.0 μM, respectively. On the other hand, l-tryptophan inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 1.0 μM, respectively. The content of syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan in the leachate of wheat straw (100 g eq./l) was 18.4 ± 0.7 and 6.2 ± 0.6 μM, respectively. Syringoylglycerol 9-O-β-d-glucopyranoside (18.4 μM) showed 21.5 and 13.5% inhibition in the lettuce and cress roots assay, respectively. On the other hand, 6.2 μM of l-tryptophan showed 47.5 and 35.0% inhibition in the lettuce and cress roots assay, respectively. These results suggested that l-tryptophan may be a major contributor to the allelopathy in aqueous leachate of wheat straw and syringoylglycerol 9-O-β-d-glucopyranoside may be a minor contributor.     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

Kinetic determination of tryptophan by using the B-Z oscillating chemical system

A simple and rapid method was devised for determination of tryptophan, based on the Belousov-Zhabotinskii (B-Z) oscillating chemical system. Changes in oscillating period and amplitude were linearly proportional to the negative logarithm of l-tryptophan concentration over the range of 6.44 × 10⁻⁷–2.55 × 10⁻⁴ M, with the regression coefficients of near unity and a lower detection limit of 6.5 × 10⁻⁸ M. d-tryptophan was also examined although it is rarely found in most biological fluids, and perhaps not at all in natural proteins. The change of period against to negative logarithm of d-tryptophan concentration over the range of 4.9 × 10⁻⁵–8.24 × 10⁻⁴ M is linear. Because the optimum conditions for determination of l- and d-tryptophan are not the same, a little amount of d-tryptophan does not affect the determination of l-tryptophan. Various influences were studied and a possible mechanism of perturbation to the B-Z oscillator by tryptophan was also discussed. Spectrophotometry and fluorescence spectrophotofluorimetry were used for comparision and confirmation of the results.     

Inhibitory action of serine on growth of bacteria of the genusBacillus on mineral synthetic media

l-Serine added to minimal synthetic media stops the growth ofBacillus subtilis, B. megaterium, B. mycoides andB. pantothenticus. All tested subspecies ofBacillus thuringiensis appear resistant to this amino acid. Serine acts bacteriostatically onB. subtilis strain B 003 and this effect depends on the concentrations of both the amino acid and the plated bacterium. Growth of serine-sensitiveBacillus strains can be restored by simultaneous addition of some other amino acids (e.g. l-threonine,l-arginine,l-aspartate orl-alanine) to the minimal media. This alleviating effect depends on the kind of amino acid. Some amino acids (e.g. l-threonine,l-tyrosine orl-tryptophan) are only effective when the serine concentration is not higher than 125 μmol/L, others (l-arginine,l-proline,l-alanine,l-aspartate orl-glutamate) are effective even when the serine concentration is as high as 500 μmol/L.     

Adsorption of a ribonucleic acid bacteriophage of Pseudomonas aeruginosa

Summary The adsorption of a ribonucleic acid bacteriophage, PP7, of Pseudomonas aeruginosa was investigated using the following approaches: electron microscopic observation of the ultrastructure of phage-pili complexes, an adsorption technique employing chloroform and rapid dilution treatment which assays the number of phages remaining unadsorbed, the effect of shear treatment on the availability of the bacterial host's adsorption sites, and the effect of metabolic inhibition of the bacterial host on phage adsorption. The specific adsorption sites on the bacterial host were found to be the pili and only when attached to the bacterial cell. Adsorption is additionally dependent upon the metabolic integrity of the host cell. Variations in the morphology of P. aeruginosa pili were observed. The mechanism of adsorption of bacteriophage PP7 appears to be similar to that of the RNA phages of two other bacterial genera, Escherichia and Caulobacter.     

Amplification of the tryptophan operon gene in Escherichia coli chromosome to increase l-tryptophan biosynthesis

Classical mutagenesis could desensitize the feedback inhibition of l-tryptophan (l-Trp) biosynthesis. Among the mutants, a5-fluorotryptophan-resistant strain, Escherichia coli EMS4-C25 produced 3 g/l of l-Trp within 18 h. The feedback-resistant l-Trp operon gene (trp) prepared from E. coli EMS4-C25 was inserted into pUC19 and pHSG576 to generate pTC701 and pTC576, respectively. When pHSG576 and pTC701 were introduced into E. coli EMS4-C25, chromosomal integration occured through homologous recombination. By using Souther hybridization, we demostrated that the integrated plasmids existed as multicopies. The strains with integrated foreign trp operon gene had higher activities of anthranilate synthase and Trp synthase than those found for the host strain and produced 9.2 g/l of l-Trp with 13% conversion yield from d-glucose. The integration and implification of the trp-operon-beraing plasmid avoided the plasmid instability and increased l-TRp production. Correspondence to: E.-C. Chan     

Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana

 The tryptophan auxotroph mutant trp3-1 of Arabidopsis thaliana (L.) Heynh., despite having reduced levels of l-tryptophan, accumulates the tryptophan-derived glucosinolate, glucobrassicin and, thus, does not appear to be tryptophan-limited. However, due to the block in tryptophan synthase, the mutant hyperaccumulates the precursor indole-3-glycerophosphate (up to 10 mg per g FW). Instability of indole-3-glycerophosphate leads to release of indole-3-acetic acid (IAA) from this metabolite during standard workup of samples for determination of conjugated IAA. The apparent increase in “conjugated IAA” in trp3-1 mutant plants can be traced back entirely to indole-3-glycerophosphate degradation. Thus, the levels of neither free IAA nor conjugated IAA increase detectably in the trp3-1 mutant compared to wild-type plants. Precursor-feeding experiments to shoots of sterile-grown wild-type plants using [²H]₅-l-tryptophan have shown incorporation of label from this precursor into indole-3-acetonitrile and indole-3-acetic acid with very little isotope dilution. It is concluded that Arabidopsis thaliana shoots synthesize IAA from l-tryptophan and that the non-tryptophan pathway is probably an artifact. Received: 1 March 2000 / Accepted: 10 April 2000     

l-Tryptophan Production by 5-Methyltryptophan-resistant Mutants of Glutamate-producing Bacteria

1. Some of 5-methyltrypotophan (5MT)-resistant mutants derived from glutamate-producing bacteria such as Brevibacterium flavum, Corynebacterium acetoglutamicum and Micrococcus glutamicus produced a small amount of l-tryptophan, while tyrosine and phenylalanine auxotrophs of B. flavum did not.

2. 5-MT-resistant mutant derived from the auxotroph for tyrosine and phenylalanine produced 390 mg/liter of l-tryptophan at most. A mutant resistant to a higher concentration of 5MT, which was derived from a tyrosine and phenylalanine auxotrophic mutant which was resistant to a low concentration of 5MT, produced 660 mg/liter of l-tryptophan. Using this mutant, the effects of the concentrations of components of the culture medium on the l-tryptophan production were examined. The high concentration of l-tyrosine, but not l-phenylalanine, inhibited the l-tryptophan production. Using the improved culture medium, this strain produced 1.9 g/liter of l-tryptophan.     

Characteristics of amino acid uptake in barley

Plants have the ability to take up organic nitrogen (N) but this has not been thoroughly studied in agricultural plants. A critical question is whether agricultural plants can acquire amino acids in a soil ecosystem. The aim of this study was to characterize amino acid uptake capacity in barley (Hordeum vulgare L.) from a mixture of amino acids at concentrations relevant to field conditions. Amino acids in soil solution under barley were collected in microlysimeters. The recorded amino acid composition, 0–8.2 μM of l-Serine, l-Glutamic acid, Glycine, l-Arginine and l-Alanine, was then used as a template for uptake studies in hydroponically grown barley plants. Amino acid uptake during 2 h was studied at initial concentrations of 2–25 μM amino acids and recorded as amino acid disappearance from the incubation solution, analysed with HPLC. The uptake was verified in control experiments using several other techniques. Uptake of all five amino acids occurred at 2 μM and below. The concentration dependency of the uptake rate could be described by Michaelis–Menten kinetics. The affinity constant (K _m) was in the range 19.6–33.2 μM. These K _m values are comparable to reported values for soil micro-organisms.     

Direct and decarboxylation-dependent effects of neurotransmitter precursors on firing of isolated monoaminergic neurons

To elucidate mechanisms that underlie the profound physiological effects of the monoamine precursors 5-hydroxy-l-tryptophan (5-HTP) and l-3,4-dihydroxyphenylalanine (l-DOPA), we examined their action on single monoaminergic neurons isolated from the ganglia of the gastropod snail Lymnaea stagnalis. In isolated serotonergic PeA motoneurons, 5-HTP produced excitation. The effect was mimicked by serotonin at 0.5–1 μM, masked by pretreatment with serotonin at higher concentrations, and abolished by the inhibitor of aromatic amino acid decarboxylase (AAAD) m-hydroxybenzylhydrazine (NSD-1015), the inhibitor of the vesicular monoamine transporter reserpine or the serotonin receptor antagonist mianserin. Exposure of the dopaminergic interneurons RPeD1 to l-DOPA caused a biphasic effect composed of a depolarization followed by a hyperpolarization. AAAD inactivation with NSD-1015, as well as the blockade of dopamine receptors with sulpiride, resulted in the enhancement of the excitatory effect, and the abolition of the inhibitory effect. Dopamine caused hyperpolarization and masked the inhibitory phase of l-DOPA action. The results show that precursors affect the rate of firing of isolated monoaminergic neurons and that their effect is completely or partially mediated by the enhanced synthesis of the respective neurotransmitter, followed by extrasynaptic release of the latter and activation of extrasynaptic autoreceptors.     

An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extremehalophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by l-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, l-tryptophan inhibited activity while l-tyrosine, l-methionine, l-leucine, and l-isoleucine activated the enzyme. l-Isoleucine and l-phenylalanine were effective at M levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by -2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by l-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by l-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

From scratch to value: engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> wild type cells to the production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine and other fine chemicals derived from chorismate

Recombinant strains of Escherichia coli K-12 for the production of the three aromatic amino acids (l-phenylalanine, l-tryptophan, l-tyrosine) have been constructed. The largest demand is for l-phenylalanine (l-Phe), as it can be used as a building block for the low-calorie sweetener, aspartame. Besides l-Phe, an increasing number of shikimic acid pathway intermediates can be produced from appropriate E. coli mutants with blocks in this pathway. The last common intermediate, chorismate, in E. coli not only serves for production of aromatic amino acids but can also be used for high-titer production of non-aromatic compounds, e.g., cyclohexadiene-transdiols. In an approach to diversity-oriented metabolic engineering (metabolic grafting), platform strains with increased flux through the general aromatic pathway were created by suitable gene deletions, additions, or rearrangements. Examples for rational strain constructions for l-phenylalanine and chorismate derivatives are given with emphasis on genetic engineering. As a result, l-phenylalanine producers are available, which were derived through several defined steps from E. coli K-12 wild type. These mutant strains showed l-phenylalanine titers of up to 38 g/l of l-phenylalanine (and up to 45.5 g/l using in situ product recovery). Likewise, two cyclohexadiene-transdiols could be recovered.     

Eco-Efficiency Analysis of biotechnological processes

For almost 50 years now, biotechnological production processes have been used for industrial production of amino acids. Market development has been particularly dynamic for the flavor-enhancer glutamate and the animal feed amino acids l-lysine, l-threonine, and l-tryptophan, which are produced by fermentation processes using high-performance strains of Corynebacterium glutamicum and Escherichia coli from sugar sources such as molasses, sucrose, or glucose. But the market for amino acids in synthesis is also becoming increasingly important, with annual growth rates of 5–7%. The use of enzymes and whole cell biocatalysts has proven particularly valuable in production of both proteinogenic and nonproteinogenic l-amino acids, d-amino acids, and enantiomerically pure amino acid derivatives, which are of great interest as building blocks for active ingredients that are applied as pharmaceuticals, cosmetics, and agricultural products. Nutrition and health will continue to be the driving forces for exploiting the potential of microorganisms, and possibly also of suitable plants, to arrive at even more efficient processes for amino acid production.     

Transport ofl-tryptophan inSaccharomyces cerevisiae

In addition to the general amino acid transport system (GAP) ofS. cerevisiae l-tryptophan is transported by another system with approximately 25% capacity of GAP, with aK _T of 0.41±0.08 mmol/L and with a similar specificity as GAP (lower inhibition by Met, Pro, Ser, Thr and 2-aminoisobutyric acid; greater inhibition by Glu and His). The pH optimum of this system is at 5.0–5.5, activation energy above the transition point (20°C) was 20 kJ/mol, below the transition point 55 kJ/mol. The transport by this system was virtually unidirectional, efflux amounting to at most 10% into a tryptophan-free medium. The transport itself was blocked by 2,4-dinitrophenol, antimycin A and uranyl nitrate. The system was synthesized de novo during preincubation with glucose=fructose>trehalose >ethanol within 30 min, and was degraded with a half-time of 15 min in the absence of further synthesis. The accumulation ratios ofl-tryptophan ingap1 mutants were concentration-dependent (200∶1 at 1 μmoll-Trp/L, 4∶1 at 2.5 mmoll-Trp/L) and decreased with increasing suspension density from 200∶1 to 5∶1 (for 10 μmoll-Trp/L). The involvement of hydrogen ions in the uptake was clearly demonstrated by the effect of D₂O even if it could not be established by either shifts of pH_out or membrane depolarization.