Regulation of Tryptophan Biosynthetic Enzymes in Neurospora crassa

The formation of enzymatic activities involved in the biosynthesis of tryptophan in Neurospora crassa was examined under various conditions in several strains. With growth-limiting tryptophan, the formation of four enzymatic activities, anthranilic acid synthetase (AAS), anthranilate-5-phosphoribosylpyrophosphate phosphoribosyl transferase (PRAT), indoleglycerol phosphate synthetase (InGPS), and tryptophan synthetase (TS) did not occur coordinately. AAS and TS activities began to increase immediately, whereas PRAT and InGPS activities began to increase only after 6 to 12 hr of incubation. In the presence of amitrole (3-amino-1,2,4-triazole), the formation of TS activity in a wild-type strain was more greatly enhanced than were AAS and InGPS activities. With a tr-3 mutant, which ordinarily exhibits an elevated TS activity, amitrole did not produce an increase in TS activity greater than that observed on limiting tryptophan. With tr-3 mutants, the increased levels of TS activity could be correlated with the accumulation of indoleglycerol in the medium; prior genetic blocks which prevented or reduced the synthesis of indoleglycerol also reduced the formation of TS activity. The addition of indoleglycerol to cultures of a double mutant (tr-1, tr-3) which could not synthesize indoleglycerol markedly stimulated the production of TS activity but not PRAT activity; the production of TS activity reached the same level with limiting or with excess tryptophan. A model explaining these and other related observations on enzyme formation in N. crassa is proposed.     

End-Product Regulation of the Tryptophan-Nicotinic Acid Pathway in Neurospora crassa

The regulation of the tryptophan-nicotinic acid pathway in Neurospora crassa was examined with mutants (nic-2, nic-3) which require nicotinamide for growth. The accumulation of N-acetylkynurenin and 3-hydroxyanthranilic acid by these mutants served to estimate the level of function of the early reactions in the pathway. In still cultures, maximal accumulation occurred with media containing growth-limiting amounts of nicotinamide; the accumulation of intermediates was almost negligible with nicotinamide in excess. Only nicotinamide and closely related compounds which also supported the growth of these mutants inhibited the accumulation of intermediates. The site of inhibition was assessed to be between tryptophan and kynurenin (or N-acetylkynurenin). The synthesis of N-acetylkynurenin was examined in washed germinated conidia suspended in buffer; the level of N-acetylkynurenin-synthesizing activity was inversely related to the concentration of nicotinamide in the germination medium. The addition of large amounts of nicotinamide to suspensions of germinated conidia did not affect their N-acetylkynurenin-synthesizing activity. Formamidase activity, kynurenin-acetylating activity, and gross tryptophan metabolism in germinated conidia was not influenced by the concentration of nicotinamide in the germination medium. The results obtained indicate that the site of inhibition by nicotinamide is the first step in the pathway, the tryptophan pyrrolase reaction. The data are interpreted as nicotinamide or a product thereof, such as nicotinamide adenine dinucleotide, acting as a repressor of the formation of tryptophan pyrrolase in N. crassa.     

Tryptophan catabolism during sporulation in Bacillus cereus

1. Two intermediates of tryptophan catabolism were isolated from a sporulating culture of Bacillus cereus and identified as anthranilic acid and kynurenine by their spectral properties. 2. During sporulation the rate of formation of anthranilic acid and kynurenine by whole cells increased and reached a maximum at the pre-spore stage. 3. The specific activities of tryptophan pyrrolase and formylase also increased during sporulation and exhibited a maximal activity at the pre-spore stage. 4. Kynureninase activity reached a maximum during early stages of sporulation and then started to decline. 5. There was a net increase in the activity of tryptophan pyrrolase when cells were grown in the presence of l-tryptophan or dl-kynurenine. 6. The cultures exhibited the maximal activity of kynureninase 2h earlier in the presence of dl-kynurenine whereas l-tryptophan delayed the appearance of the maximal activity by 2h. 7. The omission of glucose from the medium had no effect on the pattern of development of tryptophan pyrrolase during growth and sporulation. 8. On the addition of tryptophan to a chemically defined medium no significant change in the pattern of development of tryptophan pyrrolase was observed.     

Genetic control of amino acid permeability in Neurospora crassa

Lester, Gabriel (Reed College, Portland, Ore.). Genetic control of amino acid permeability in Neurospora crassa. J. Bacteriol. 91:677-684. 1966.-Strains of Neurospora crassa resistant to 4-methyltryptophan (4-MT) were isolated from populations of conidia exposed to ultraviolet light. In genetic crosses, 4-MT resistance behaved as a single-gene difference. Resistance to 4-MT could not be attributed to a relaxation of control of the formation or the activity of the enzymes of tryptophan biosynthesis. Growth studies involving tryptophan auxotrophs carrying the aberrant mt gene and uptake studies with normal and 4-MT-resistant strains showed that 4-MT resistance could be attributed to an inability of 4-MT-resistant strains to take up tryptophan and its methyl analogues. The mt gene is not specific for tryptophan; strains resistant to 4-MT are also resistant to ethionine, and they have a markedly reduced ability to take up serine, leucine, and alpha-aminoisobutyric acid. No difference was observed between strains carrying either mt allele in their ability to take up glucose; also, the uptake of anthranilic acid or of indole was not sufficiently impaired by the aberrant mt gene to prevent these tryptophan precursors from satisfying the nutritional requirement of certain tryptophan auxotrophs. The role of the mt gene in determining the permeability of N. crassa to amino acids is discussed.     

L-tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo

The administration of l-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo. When l-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy-dl-tryptophan and indole administration in amounts equimolar to l-tryptophan also result in similar increases in activity whereas α-methyl-dl-tryptophan produces little or no increase.Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.     

AMINO ACID INTERACTIONS IN NEUROSPORA CRASSA.

Soboren, Josephine (University of California, Los Angeles), and Joseph F. Nyc. Amino acid interactions in Neurospora crassa. J. Bacteriol. 82:20-25. 1961.-A systematic study of the effects of the naturally occurring amino acids on the growth of a wild-type strain of Neurospora crassa focused attention upon l-tryptophan, which exhibits a strong growth inhibitory effect. Further investigation disclosed that other tryptophan metabolites, anthranilic acid, indole, kynurenine, and 3-hydroxykynurenine also inhibit growth. The proposed antimetabolic role of these aromatic compounds explains the poor growth response of certain tryptophan-requiring strains of N. crassa to tryptophan supplements. The growth of normal and mutant strains of N. crassa on media supplemented with tryptophan is influenced by the presence of other amino acids.     

Indole-3-acetic acid (IAA) synthesis in the biocontrol strain CHA0 of Pseudomonas fluorescens: role of tryptophan side chain oxidase.

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent against soil-borne fungal plant pathogens. In this study, indole-3-acetic acid (IAA) biosynthesis in strain CHA0 was investigated. Two key enzyme activities were found to be involved: tryptophan side chain oxidase (TSO) and tryptophan transaminase. TSO was induced in the stationary growth phase. By fractionation of a cell extract of strain CHA0 on DEAE-Sepharose, two distinct peaks of constitutive tryptophan transaminase activity were detected. A pathway leading from tryptophan to IAA via indole-3-acetamide, which occurs in Pseudomonas syringae subsp. savastanoi, was not present in strain CHA0. IAA synthesis accounted for less than or equal to 1.5% of exogenous tryptophan consumed by resting cells of strain CHA0, indicating that the bulk of tryptophan was catabolized via yet another pathway involving anthranilic acid as an intermediate. Strain CHA750, a mutant lacking TSO activity, was obtained after Tn5 mutagenesis of strain CHA0. In liquid cultures (pH 6.8) supplemented with 10 mM-L-tryptophan, growing cells of strains CHA0 and CHA750 synthesized the same amount of IAA, presumably using the tryptophan transaminase pathway. In contrast, resting cells of strain CHA750 produced five times less IAA in a buffer (pH 6.0) containing 1 mM-L-tryptophan than did resting cells of the wild-type, illustrating the major contribution of TSO to IAA synthesis under these conditions. In artificial soils at pH approximately 7 or pH approximately 6, both strains had similar abilities to suppress take-all disease of wheat or black root rot of tobacco. This suggests that TSO-dependent IAA synthesis is not essential for disease suppression.     

Inhibition of Growth, Synthesis, and Permeability in Neurospora crassa by Phenethyl Alcohol.

Lester, Gabriel (Reed College, Portland, Ore.). Inhibition of growth, synthesis, and permeability in Neurospora crassa by phenethyl alcohol. J. Bacteriol. 90: 29-37. 1965.-Inhibition of the growth of Neurospora crassa in still culture was detected at 0.05% and was complete at a level of 0.2% phenethyl alcohol (PEA). Benzyl alcohol was less inhibitory, and 3-phenyl-1-propanol and phenol were more inhibitory, than PEA; benzylamine and phenethylamine were less inhibitory than the analogous hydroxylated compounds. Inhibition by PEA was not reversed by synthetic mixtures of purines and pyrimidines or vitamins, or by casein digests, yeast extract, or nutrient broth. The germination of conidia was inhibited by PEA, but after an exposure of 8.5 hr no loss of viability was observed. The addition of PEA to growing shake cultures caused a simultaneous inhibition of growth and of the syntheses of ribonucleic and deoxyribonucleic acids and protein; the relationships of these compounds to mycelial dry weight and to one another were constant in growing mycelia, and PEA did not significantly affect these relationships. PEA partially inhibited the uptake of glucose, but severely restricted the accumulation of l-leucine, l-tryptophan, or alpha-aminoisobutyric acid in germinated conidia. The efflux of alpha-aminoisobutyric acid from germinated conidia was somewhat enhanced by PEA, but this effect was not so pronounced as the (complete) inhibition of alpha-aminoisobutyric acid accumulation by PEA. It is suggested that PEA affects primarily the initial influx of alpha-aminoisobutyric acid rather than the subsequent retention of alpha-aminoisobutyric acid.     

Microbiological method for the determination of L-tryptophan

Sebek, Oldrich K. (The Upjohn Company, Kalamazoo, Mich.). Microbiological method for the determination of l-tryptophan. J. Bacteriol. 90:1026-1031. 1965.-The ability of Chrombacterium violaceum to utilize l-tryptophan for the synthesis of a purple pigment, violacein, served as a basis for the development of a quantitative estimation of this amino acid. The method consists of suspending washed colorless cells of the organism in an agar layer, placing a paper disc impregnated with a tryptophan solution on top of the layer, and allowing the system to incubate. As tryptophan diffuses into the agar, it is converted into violacein, and appears as a zone of striking purple color. Since the diameter of the zone is a function of the amount of tryptophan applied, the amino acid can be quantitatively estimated within the range of 10 to 320 mug per sample with 5.6% standard deviation. The method is fairly specific for free tryptophan, since only indole, indole-3-pyruvic acid, and, to a small degree, anthranilic acid interfere. Other amino acids, tissue homogenates, tryptophan in peptide linkage, or compounds related to this amino acid do not affect its determination. The bacterium does not utilize tryptophan for the synthesis of cellular material unless its growth has been initiated by another substrate.     

Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Insights from quantum chemistry.

Quantum chemical methods AM1 and PM3 and chromatographic methods were used to qualitatively characterize pathways of bacterial production of indole-3-acetic acid (IAA). The standard free energy changes (delta G(o)'sum) for the synthesis of tryptophan (Trp) from chorismic acid via anthranilic acid and indole were calculated, as were those for several possible pathways for the synthesis of IAA from Trp, namely via indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), and indole-3-acetonitrile (IAN). The delta G(o)'sum for Trp synthesis from chorismic acid was -402 (-434) kJ.mol-1 (values in parentheses were calculated by PM3). The delta G(o)'sum for IAA synthesis from Trp were -565 (-548) kJ.mol-1 for the IAN pathway, -481 (-506) kJ.mol-1 for the IAM pathway, and -289 (-306) kJ.mol-1 for the IPyA pathway. By HPLC analysis, the possibility was assessed that indole, anthranilic acid, and Trp might be utilized as precursors for IAA synthesis by Azospirillum brasilense strain Sp 245. The results indicate that there is a high motive force for Trp synthesis from chorismic acid and for IAA synthesis from Trp, and make it unlikely that anthranilic acid and indole act as the precursors to IAA in a Trp-independent pathway.     

Production of Substituted l-Tryptophans by Fermentation

Claviceps purpurea has been shown to produce extracellular l-tryptophan from indole in stirred fermentors. The substrate specificity of this conversion was investigated by using substituted indoles, anthranilic acid, and 4-chloro-anthranilic acid. Addition of 2-, 4-, 5-, 6-, and 7-methyl indole or 6-chloroindole to C. purpurea C1M produced the corresponding substituted l-tryptophan. In contrast, addition of l-methyl, 6-trifluoromethyl, 6-nitro-, or 4-benzyloxy-substituted indoles, or anthranilic and 4-chloroanthranilic acids did not produce detectable amounts of the corresponding tryptophan.     

Alteration of Tryptophan-mediated Regulation in Neurospora crassa by Indoleglycerol Phosphate

The accumulation of imidazoleglycerol phosphate during growth of Neurospora crassa in the presence of 3-amino-1,2,4-triazole was found to cause derepression of tryptophan synthetase and to inhibit the induction of kynureninase. Accumulation of indoleglycerol phosphate in response to growth in the presence of indole acrylic acid or anthranilic acid was also accompanied by derepressed synthesis of tryptophan synthetase. Enzyme synthesis in mutants (his-7 and trp-4) unable to form these intermediates was not altered under similar conditions. The rate of formation of tryptophan synthetase and kynureninase was found to differ in the presence of tryptophan and indole.     

Metabolism of Tryptophan, Indole-3-acetic Acid, and Related Compounds in Parasitic Plants from the Genus Orobanche

Metabolic reactions involving the aliphatic side chain of tryptophan were studied in the holoparasitic dicotyledonous plants Orobanche gracilis Sm., O. lutea Baumg., and O. ramosa L. Unlike known autotrophic plants, the parasite metabolized l-tryptophan directly to indole-3-carboxaldehyde, which was further converted to indole-3-methanol and indole-3-carboxylic acid. Independently, these metabolites were also formed from d-tryptophan, tryptamine, indole-3-lactic acid, and indole-3-acetic acid. As in autotrophic plants, tryptophan and tryptamine were also converted, via indole-3-acetaldehyde, to indole-3-acetic acid, indole-3-ethanol, and its glucoside. The branch of tryptophan metabolism relevant to auxin biogenesis and catabolism is, therefore, not rudimentary in Orobanche but even more complex than in autotrophic higher plants.     

The effect of pyrazines on the metabolism of tryptophan and nicotinamide adenine dinucleotide in the rat Evidence of the formation of a potent inhibitor of aminocarboxy-muconate-semialdehyde decarboxylase from pyrazinamide

The intraperitoneal or oral administration of pyrazinamide and pyrazinoic acid (pyrazine 2-carboxylic acid) resulted in a marked increase of the NAD content in rat liver. The injections of pyrazine and pyrazine 2,3-dicarboxylic acid exhibited no significant effect on the hepatic NAD content. The boiled extract obtained from liver and kidney of rat injected with either pyrazinamide or pyrazinoic acid exhibited a potent inhibitory effect on the aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) activity in either liver or kidney, although pyrazinamide or pyrazinoic acid per se did not inhibit the enzyme activity. The unknown inhibitor of aminocarboxymuconate-semialdehyde decarboxylase was dialysable and heat-stable, and mostly excreted in urine by 6 and 12 h after injection of pyrazinoic acid and pyrazinamide, respectively. Pyrazine 2,3-dicarboxylic acid, pyrazine, nicotinamide, nicotinic acid, tryptophan, anthranilic acid, 5-hydroxyanthranilic acid and quinolinic acid exhibited no significant effect on the aminocarboxymuconate-semialdehyde decarboxylase activity in liver and kidney at the concentration of 1 mM in the reaction mixture. The expired ¹⁴CO₂ from l-[benzen ring-U-¹⁴C]tryptophan was markedly decreased by the pyrazinamide injection, while the urinary excretion of ¹⁴C-labeled metabolites from l-tryptophan, mainly quinolinic acid, was markedly increased. These results suggest that the glutarate pathway of l-tryptophan was strongly inhibited by the inhibitor produced after the administration of pyrazinoic acid and pyrazinamide. Pyrazinamide but not pyrazinoic acid also exhibited a significant inhibition of the nuclear enzyme poly(ADP-ribose) synthetase in rat liver.     

Preparation and analysis of mutants of rhizospheric pseudomonads, resistant to toxic analogs of tryptophan and phenylalanine]

The absence of plasmids in strains of fluorescent pseudomonads characterized by high level of synthesis of phytohormone indole-3-acetic acid (IAA) as well as invariability of this feature in plasmid and non-plasmid variants of strain BSP8 suggests chromosomal control of IAA synthesis by the rhizosphere bacteria tested. Using toxic analogues of aromatic amino acids -5-fluorine-tryptophan and 5-methyl-tryptophan variants were obtained which synthesized and secreted only anthranilic acid. Mutants with resistance to p-fluorine-phenylalanine and capable of secreting tryptophan and/or phenylalanine were found. Testing of the secreting variants failed to reveal any differences between the levels of IAA biosynthesis in comparison with the wild-type strains.     

Biosynthesis of glyantrypine by Aspergillus clavatus

The biosynthesis of glyantrypine from radiolabelled amino acid precursors has been shown experimentally to involve anthranilic acid, tryptophan and glycine. Low values for percentage incorporation of radiolabel into glyantrypine were partly influenced by a complex array of other novel alkaloids shown by the radiolabelling experiments to be related to glyantrypine. Interpretation of radiolabel incorporation from [14C-carboxyl]-anthranilic acid into microbial metabolites seen to contain an anthranilyl moiety in various biosynthetic arrangements is discussed. The possibility of diversion of anthranilic acid from the kynurenine pathway to glyantrypine biosynthesis is recognised.     

模块化工程改造大肠杆菌生产L-色氨酸

L-色氨酸作为一种必需氨基酸,广泛应用于食品、饲料和医药等领域。目前,微生物法生产L-色氨酸存在转化率低等问题。为此,本研究通过敲除L-色氨酸操纵子阻遏蛋白(L-tryptophan operon repressor protein, trpR)、替换l-色氨酸弱化子(trpL)、引入抗反馈调节的aroG^fbr等,获得可积累11.80 g/L L-色氨酸的底盘菌株大肠杆菌(Escherichia coli)TRP3。在此基础上,将L-色氨酸合成途径分为中心代谢途径模块、莽草酸(shikimic acid, SA)途径至分支酸(chorismic acid, CHA)模块、分支酸至L-色氨酸模块,并借助启动子工程,通过平衡中心代谢途径模块、莽草酸途径至分支酸模块、分支酸至L-色氨酸模块,获得工程菌E.coli TRP9。在5 L发酵罐中,工程菌E.coli TRP9的L-色氨酸产量提升至36.08 g/L,糖酸转化率提升至18.55%,达到理论转化率的81.7%。本研究利用模块工程策略,构建了高产L-色氨酸生产菌株,为l-色氨酸的规模化生产奠定了良好的基础。     

In planta production of indole-3-acetic acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.     

Indole-3-Acetic Acid Biosynthesis in Colletotrichum gloeosporioides f. sp. aeschynomene

We characterized the biosynthesis of indole-3-acetic acid by the mycoherbicide Colletotrichum gloeosporioides f. sp. aeschynomene. Auxin production was tryptophan dependent. Compounds from the indole-3-acetamide and indole-3-pyruvic acid pathways were detected in culture filtrates. Feeding experiments and in vitro assay confirmed the presence of both pathways. Indole-3-acetamide was the major pathway utilized by the fungus to produce indole-3-acetic acid in culture.