Photoproduction of amino acids by mutant strains of N2-fixing cyanobacteria

Summary Mutant strains of the N₂-fixing cyanobacterium bacterium Anabaena variabilis resistant to 6-fluorotryptophan or to ethionine were isolated. Many of these strains liberated amino acids into their media in the absence of 6-fluorotryptophan and ethionine. Nitrogenase activity was higher in mutant strains than in the parent strain. Mutant strains were immobilised in calcium alginate and sustained photoproduction of amino acids has been demonstrated.Abbreviations ETH ethionine - FT 6-fluorotryptophan - Hepes 4-(2-hydroxyethyl)-1, piperazine ethanesulphonic acid - PEP phosphoenolpyruvate - DAHP 3-deoxy-d-arabinoheptulosonate 7-phosphate - chl a chlorophyll a     

Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239

The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenylalanine and tyrosine, whereas prephenate dehydratase was very sensitive to inhibition by phenylalanine. Tyrosine was a strong activator of the latter enzyme, whereas anthranilate synthase was inhibited effectively by tryptophan. No clear repression of the synthesis of these enzymes was observed during growth of the organism in the presence of the aromatic amino acids. It is therefore concluded that in Nocardia sp. 239 synthesis of these amino acids is mainly regulated by feedback inhibition. The molecular organization and kinetic properties of DAHP synthase were studied in more detail following its purification. The molecular weight of the native enzyme and its single subunit species were estimated to be 168,000 and 41,000, respectively, suggesting that the enzyme is a tetramer. Apparent K _m values for phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) were 45 and 370 M, respectively. Tryptophan, phenylalanine and tyrosine inhibited DAHP synthase in a competitive manner with respect to E4P, with apparent K _i values of 3, 160 and 180 M, respectively. In addition, tryptophan and E4P (apparent K _i values of 11 and 530 M, respectively) were found to exert an uncompetitive and competitive inhibition, respectively, towards PEP.Abbreviations DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate - E4P erythrose-4-phosphate - PEP phosphoenolpyruvate - RuMP ribulose monophosphate - HPLC high performance liquid chromatography - FPLC fast protein liquid chromatography - SDS sodium dodecyl sulphate     

De-regulated assimilation and over-production of amino acids in analogue-resistant mutants of a cyanobacterium,Phormidium uncinatum

Mutant strains of Phormidium uncinatum resistant to fluoro-phenylalanine, aztryptophan, fluorotyrosine and azaleucine accumulated a wide range of amino acids, notably glutamic acid, lysine, tyrosine and phenylalanine, and exhibited de-regulated valine and phenylalanine transport. While acetohydroxy acid synthase in azaleucine-resistant mutants lost valine- and leucine-sensitivity, 3-deoxy-Dxxx-arabinoheplulosonate-7-phosphate (DAHP) synthase and prephenate dehydratase in aromatic analogue-resistant strains became phenylalanine-insensitive and shikimate and prephenate dehydrogenases were activated by tyrosine. In addition, activities of nitrate-assimilating enzymes were higher in the mutants, which also exhibited increased nitrogen, protein and phycocyanin contents. The proteins in the mutants were better digested upon enzymatic-treatments and feeding trials than those of the wild type, indicating that they are usable as single-cell protein.N.S. Rao and T.M. Shakila are and S.N. Bagchi was with the Department of Biological Sciences, R.D. University, Jabalpur-482 001, Madhya Pradesh, India. S.N. Bagchi is now with the Department of Microbiology, MDS University, Amjer-305 001, Rajasthan, India.     

Enzymic arrangement and allosteric regulation of the aromatic amino acid pathway in Neisseria gonorrhoeae

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by l-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by l-phenylalanine. Prephenate dehydratase was strongly activated by l-tyrosine. NAD⁺-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by l-tyrosine. Two aromatic aminotransferases were resolved, one preferring the l-phenylalanine:2-ketoglutarate substrate combination and the other preferring the l-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which l-tyrosine modulates l-phenylalanine synthesis via activation of prephenate dehydratase. l-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.     

The stable phylogenetic distribution of the recently evolvedl-phenylalanine-inhibited isozyme of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in enteric bacteria

Escherichia coli and some other enteric bacteria possess three regulatory isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, each of which is inhibited by one aromatic amino acid. Thel-phenylalanine-sensitive isozyme of DAHP synthase has evolved most recently since it is absent in all other members of the Gram-negative cluster that contains enteric bacteria as a subcluster. A comprehensive survey of enteric genera was carried out to determine whether the newly evolved isozyme is a stable, conserved trait. The results obtained show that all the genera of the contemporaryEnterobacteriaceae family possess the recently evolved phenylalanine-sensitive isozyme in addition to the tyrosine-and tryptophansensitive isozymes of DAHP synthase. However, physiological manipulation was usually necessary to derepress the tryptophan-sensitive DAHP synthase in order to demonstrate its presence.Florida Experiment Station Journal Series No. 9603.     

The Herbicidally Active Compound N-2-(6-Methyl-Pyridyl)-Aminomethylene Bisphosphonic Acid Inhibits In Vivo Aromatic Biosynthesis

The effect of N-2-(6-methyl-pyridyl)-aminomethylene bisphosphonic acid (M-pyr-AMBPA), a compound previously shown to exhibit herbicidal properties on whole plants and to inhibit in vitro activity of the first enzyme in the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase, was investigated on Nicotiana plumbaginifolia suspension cultured cells and compared to that of the herbicide glyphosate. The addition of M-pyr-AMBPA from 10⁻⁴ to 10⁻³ M was found to cause a severe cell growth reduction. Kinetic analysis of partially purified DAHP synthase accounted for non-competitive inhibition type with respect to both phospho-enol-pyruvate and erythrose-4-phosphate, with K_I values of 0.43 and 0.62 mM, respectively. Amino acid pool measurements of cells grown in the presence of sublethal doses of M-pyr-AMBPA pointed to an actual reduction of free aromatic amino acids, showing that DAHP synthase inhibition takes place in vivo, and suggesting that the interference of this aminophosphonate with plant aromatic biosynthesis may account for a large part of its phytotoxicity. However, exogenous supply of a mixture of phenylalanine, tyrosine and tryptophan failed to achieve full reversal of cell growth inhibition, yet the occurrence of other target(s) cannot be ruled out. Received November 24, 1998; accepted June 3, 1999     

Isolation of cyanobacterial auxotrophs by direct selection of regulatory-mutant phenotypes

We have isolated a chorismate mutase bradytroph (leaky auxotroph) ofAnabaena sp. PCC 7119 (ATCC 29151) as a spontaneous 6-fluorotryptophan-resistant mutant. The decreased chorismate mutase activity resulted in the production of quantities of the phenylalanine and tyrosine that limited rate of growth. 3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity in the mutant was elevated more than twofold over the wild-type activity, suggesting derepression of this enzyme. The physiological deregulation of DAHP synthase and the genetic-based deficiency of chorismate mutase promoted an elevated level of intracellular chorismate, which then overwhelmed the competitive inhibition of anthranilate synthase by tryptophan, resulting in the overproduction of tryptophan and indoleglycerolphosphate. The presence of exogenous serine increased the production of tryptophan at the expense of indoleglycerolphosphate. This indicated that the endogenous potential for increasing the amount of serine available for increased tryptophan production is limited.     

Phenylalanine hydroxylase and isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in relationship to the phylogenetic position of Pseudomonas acidovorans (Ps. sp. ATCC 11299a)

The evolution of aromatic amino acid biosynthesis and its regulation is under study in a large assemblage of prokaryotes (Superfamily A) whose phylogenetic arrangement has been constructed on the criterion of oligonucleotide cataloging. One section of this Superfamily consists of a well defined (rRNA homology) cluster denoted as Group III pseudomonads. Pseudomonas acidovorans ATCC 11299a, a Group III member, was chosen for indepth studies of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase, the initial regulatory enzyme of aromatic biosynthesis. This strain is of particular interest for evolutionary studies of aromatic metabolism because it possesses phenylalanine hydroxylase, an enzyme whose physiological role and distribution among prokaryotes is largely unknown. Although P. acidovorans ATCC 11299a has been of uncertain identity, we now establish it unambiguously as a species of acidovorans by virtue of its 87% DNA homology with P. acidovorans ATCC 15668 (type strain). This result conformed with enzyme patterning studies which placed ATCC 11299a into pseudomonad Group IIIa, a subgroup containing the acidovorans species. Crude extracts of Group III pseudomonads had previously been shown to share, as a common group characteristic, sensitivity of DAHP synthase to feedback inhibition by either l-tyrosine or l-phenylalanine. Detailed studies with partially purified preparations from strain ATCC 11299a revealed the presence of two distinct regulatory isozymes, DAHP synthase-phe and DAHP synthase-tyr. DAHP synthase-tyr is tightly controlled by l-tyrosine with 50% inhibition of activity being achieved at 4.0 M effector. DAHP synthase-phe is inhibited 50% by 40 M l-phenylalanine and exhibits dramatic changes in levels of activity, as well as chromatographic elution patterns, in response to dithiothreitol. This two-isozyme pattern of DAHP synthase has not been described previously, although it may prove to be widespread.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - E4P d-erythrose-4-phosphate - PEP phosphoenolpyruvate - DTT dithiothreitol - BSA fraction V bovine serum albumin     

Aromatic amino acid biosynthesis in Buchnera aphidicola (Endosymbiont of aphids): Cloning and sequencing of a DNA fragment containing aroH-thrS-infC-rpmI-rplT

A 4.5-kilobase DNA fragment from Buchnera aphidicola, the endosymbiont of the aphid Schizaphis graminum, was cloned and sequenced. On the basis of homology to Escherichia coli, the following genes were found in the order listed: aroH-thrS-infC-rpmI-rplT. AroH corresponds to the E. coli tryptophan-inhibited 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase. Evidence was presented indicating that this is the sole gene for DAHP synthase in the B. aphidicola genome. This enzyme initiates the complex branched pathway leading to aromatic amino acid biosynthesis. The presence of aroH is consistent with past observations indicating that aphid endosymbionts are able to synthesize tryptophan for the aphid host. thrS, infC, rpmI, and rplT correspond to genes for threonine tRNA synthase, initiation factor-3, and large ribosome subunit proteins L35 and L20, respectively. Sequence comparisons indicate some differences and similarities between E. coli and B. aphidicola with respect to the possible regulation of synthesis of these proteins.     

Biosynthesis of aromatic amino acids in Nocardia sp. 239: effects of amino acid analogues on growth and regulatory enzymes

Summary Further steps required for overproduction of aromatic amino acids by a mutant strain of Nocardia sp. 239 (Noc 87-13), unable to grow on l-phenylalanine as a sole carbon and energy source, were investigated. A number of analogues of the aromatic amino acids displayed severe inhibitory effects on the activities of regulatory enzymes in the biosynthetic pathway and growth of the organism in glucose mineral medium. l-Tryptophane analogues strongly inhibited 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity. l-Tyrosine analogues especially inhibited DAHP synthase and chorismate mutase, whereas l-phenylalanine analogues strongly inhibited chorismate mutase and prephenate dehydratase activity. Addition of the aromatic amino acids and their precursors chorismate, 4-hydroxyphenylpyruvate, phenylpyruvate and anthranilate, to the medium counteracted the growth inhibitory effect of specific analogues. The data indicate that ortho- (OFP) and para-fluoro-d,l-phenylalanine (PFP), and l-phenylalanine amide, are the most suitable analogues for the isolation of feedback-inhibition-insensitive prephenate dehydratase mutants. Attempts to isolate l-tyrosine and l-trytophane auxotrophic mutants were only successful in the latter case, resulting in the selection of a stable anthranilate synthase-negative mutant (Noc 87-13-14). Uptake of aromatic amino acids in Nocardia sp. 239 most likely involves a common transport system. This necessitates the use of anthranilate, rather than l-trytophane, as a supplement during the isolation of l-tyrosine auxotrophic and OFP- and/or PFP-resistant mutant derivative strains of Noc 87-13-14. Offprint requests to: L. Dijkhuizen     

Obtaining Pseudomonas aurantiaca strains capable of overproduction of phenazine antibiotics

N-methyl-N′-nitro-N-nitrosoguanidine (NG)-induced mutagenesis with subsequent selection for resistance to toxic amino acid analogues (azaserine, m-fluoro-DL-phenylalanine, and 6-diazo-5-oxo-L-norleucine) was applied to Pseudomonas aurantiaca B-162. The resulting strains produced phenazine antibiotics three times more efficiently than the wild type strain and ten times more efficiently than the known pseudomonad strains. Overproduction of phenazine antibiotics was shown to result either from deregulation of 3-deoxy-D-arabinohepulosonate-7-phosphate synthase (DAHP synthase), the key enzyme of the aromatic pathway (removal of inhibition by phenylalanine, tyrosine, and phenazine), or overproduction of N-hexanoyl homoserine lactone, the regulatory molecules of positive control of cellular metabolism (QS systems).     

Regulation of Aromatic Amino Acid Biosynthesis and Production of Tyrosine and Phenylalanine m Brevibacterium flavum

In Brevibacterium flavum, prephenate dehydratase in the phenylalanine specific biosynthetic pathway was strongly inhibited by phenylalanine and activated by tyrosine. Furthermore. the inhibition by phenylalanine was completely reversed by tyrosine. Inhibition by tyrosine of prephenate dehydrogenase in the tyrosine specific pathway was very weak. Overall regulation mechanism of the aromatic amino acid biosynthesis in B. flavum was proposed on the bases of these results and the previous findings on 3-deoxy-D-arabino-heptulosonate-7- phosphate synthetase(DAHP synthetase*) of the common pathway and on anthranilate synthetase of the tryptophan specific pathway. Two types of m-fluorophenylalanine(mFP) resistant mutants which accumulated phenylalanine alone or both phenylalanine and tyrosine, respectively, were derived. The accumulation in the former mutants was inhibited by tyrosine, but that in the latter was affected neither by tyrosine nor by phenylalanine. DAHP synthetase of the latter mutants had been desensitized from the synergistic feedback inhibition by tyrosine and phenylalanine, while prephenate dehydratase of the former mutants had been desensitized in the feedback inhibition by phenylalanine. Tyrosine auxotroph accumulated phenylalanine under tyrosine limitation and its accumulation was inhibited by the excessive addition of tyrosine. Phenylalanine auxotroph accumulated tyrosine under phenylalanine limitation and its accumulation was inhibited by the excessive addition of phenylalanine. These results in vivo strongly supported the proposed regulation mechanism in which synthesis of phenylalanine in preference to tyrosine was assumed.     

Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

Repression of aromatic amino acid biosynthesis in Escherichia coli K-12

Mutants of Escherichia coli K-12 were isolated in which the synthesis of the following, normally repressible enzymes of aromatic biosynthesis was constitutive: 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetases (phe and tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A. In the wild type, DAHP synthetase (phe) was multivalently repressed by phenylalanine plus tryptophan, whereas DAHP synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A were repressed by tyrosine. DAHP synthetase (tyr) and chorismate mutase T-prephenate dehydrogenase were also repressed by phenylalanine in high concentration (10(-3)m). Besides the constitutive synthesis of DAHP synthetase (phe), the mutants had the same phenotype as strains mutated in the tyrosine regulatory gene tyrR. The mutations causing this phenotype were cotransducible with trpA, trpE, cysB, and pyrF and mapped in the same region as tyrR at approximately 26 min on the chromosome. It is concluded that these mutations may be alleles of the tyrR gene and that synthesis of the enzymes listed above is controlled by this gene. Chorismate mutase P and prephenate dehydratase activities which are carried on a single protein were repressed by phenylalanine alone and were not controlled by tyrR. Formation of this protein is presumed to be controlled by a separate, unknown regulator gene. The heat-stable phenylalanine transaminase and two enzymes of the common aromatic pathway, 5-dehydroquinate synthetase and 5-dehydroquinase, were not repressible under the conditions studied and were not affected by tyrR. DAHP synthetase (trp) and tryptophan synthetase were repressed by tryptophan and have previously been shown to be under the control of the trpR regulatory gene. These enzymes also were unaffected by tyrR.     

Application of metabolic engineering to improve both the production and use of biotech indigo

A fermentation process was developed for production of indigo from glucose using recombinant Escherichia coli. This was achieved by modifying the tryptophan pathway to cause high-level indole production and adding the Pseudomonas putida genes encoding naphthalene dioxygenase (NDO). In comparison to a tryptophan-overproducing strain, the first indigo-producing strain made less than half of the expected amount of indigo. Severe inactivation of the first enzyme of aromatic biosynthesis, 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase (the aroG ^fbr gene product), was observed in cells collected from indigo fermentations. Subsequent in vitro experiments revealed that DAHP synthase was inactivated by exposure to the spontaneous chemical conversion of indoxyl to indigo. Indigo production was thereafter improved by increasing the gene dosage of aroG ^fbr or by increasing substrate availability to DAHP synthase in vivo by either amplifying the tktA (transketolase) gene or inactivating both isozymes of pyruvate kinase. By combining all three strategies for enhancing DAHP formation in the cell, a 60% increase in indigo production was achieved. Metabolic engineering was then further applied to eliminate a byproduct of the spontaneous conversion of indoxyl to indigo, thereby solving a serious problem with the use of bio-indigo in the final denim dyeing application. Journal of Industrial Microbiology & Biotechnology (2002) 28, 127–133 DOI: 10.1038/sj/jim/7000228 Received 20 May 2001/ Accepted in revised form 10 November 2001     

Evolution of aromatic biosynthesis and fine-tuned phylogenetic positioning of Azomonas,Azotobacter and rRNA group I pseudomonads

The evolutionary history of biochemical pathways can be determined in microbial groupings for which phylogenetic trees have been established. This has been demonstrated best in Superfamily B, an assemblage of rRNA homology groups containing lineages that lead to genera such as Escherichia and other enteric microbes, Pseudomonas (Group I), Xanthomonas, Oceanospirillum, and Acinetobacter. The rRNA homology group that defines Group I pseudomonads also includes Azomonas and Azotobacter, but particular dendrogram points of evolutionary divergence for these genera within Superfamily B have not been established. Phylogenetic relationships at such intergeneric levels can be deduced by analysis of aromaticpathway enzyme arrangement and regulation in selected groupings where dynamic evolutionary changes have occurred. A case in point is illustrated by Axomonas insignis, Azotobacter paspali, and Azotobacter vinelandii — a grouping that appears to be homogeneous with respect to the evolutionary state of the aromatic pathway. The conclusion that this phylogenetic cluster diverges from an ancestor common to pseudomonad subgroup Ia (rather than to subgroup Ib) is based upon the absence of chorismate mutase-F and arogenate dehydratase, enzymes making up a twostep pathway of phenylalanine biosynthesis that is absent in subgroup Ia, but present in subgroup Ib. Of further interest, Azomonas insignis and Azotobacter sp. were found to comprise a distinctive and recently evolved sublineage, differing from subgroup Ia species in their loss of a regulatory isozyme of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (ADHP synthase-trp) that is subject to feedback inhibition by l-tryptophan. DAHP synthase-trp is an ancient character state of Superfamily B that has been retained during the evolutionary history of most members of this Superfamily.Abbreviation DAHP 3-Deoxy-d-arabino-heptulosonate 7-phosphate     

Regulation of phenylalanine biosynthesis in Rhodotorula glutinis.

The phenylalanine biosynthetic pathway in the yeast Rhodotorula glutinis was examined, and the following results were obtained. (i) 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase in crude extracts was partially inhibited by tyrosine, tryptophan, or phenylalanine. In the presence of all three aromatic amino acids an additive pattern of enzyme inhibition was observed, suggesting the existence of three differentially regulated species of DAHP synthase. Two distinctly regulated isozymes inhibited by tyrosine or tryptophan and designated DAHP synthase-Tyr and DAHP synthase-Trp, respectively, were resolved by DEAE-Sephacel chromatography, along with a third labile activity inhibited by phenylalanine tentatively identified as DAHP synthase-Phe. The tyrosine and tryptophan isozymes were relatively stable and were inhibited 80 and 90% by 50 microM of the respective amino acids. DAHP synthase-Phe, however, proved to be an extremely labile activity, thereby preventing any detailed regulatory studies on the partially purified enzyme. (ii) Two species of chorismate mutase, designated CMI and CMII, were resolved in the same chromatographic step. The activity of CMI was inhibited by tyrosine and stimulated by tryptophan, whereas CMII appeared to be unregulated. (iii) Single species of prephenate dehydratase and phenylpyruvate aminotransferase were observed. Interestingly, the branch-point enzyme prephenate dehydratase was not inhibited by phenylalanine or affected by tyrosine, tryptophan, or both. (iv) The only site for control of phenylalanine biosynthesis appeared to be DAHP synthase-Phe. This is apparently sufficient since a spontaneous mutant, designated FP9, resistant to the growth-inhibitory phenylalanine analog p-fluorophenylalanine contained a feedback-resistant DAHP synthase-Phe and cross-fed a phenylalanine auxotroph of Bacillus subtilis.     

Clues from a halophilic methanogen about aromatic amino acid biosynthesis in archaebacteria

Extensive diversity in features of aromatic amino acid biosynthesis and regulation has become recognized in eubacteria, but almost nothing is known about the extent to which such diversity exists within the archaebacteria. Methanohalophilus mahii, a methylotrophic halophilic methanogen, was found to synthesize l-phenylalanine and l-tyrosine via phenylpyruvate and 4-hydroxyphenylpyruvate, respectively. Enzymes capable of using l-arogenate as substrate were not found. Prephenate dehydrogenase was highly sensitive to feedback inhibition by l-tyrosine and could utilize either NADP⁺ (preferred) or NAD⁺ as cosubstrate. Tyrosine-pathway dehydrogenases having the combination of narrow specificity for a cyclohexadienyl substrate but broad specificity for pyridine nucleotide cofactor have not been described before. The chorismate mutase enzyme found is a member of a class which is insensitive to allosteric control. The most noteworthy character state was prephenate dehydratase which proved to be subject to multimetabolite control by feedback inhibitor (l-phenylalanine) and allosteric activators (l-tyrosine, l-tryptophan, l-leucine, l-methionine and l-isoleucine). This interlock type of prephenate dehydratase, also known to be broadly distributed among the gram-positive lineage of the eubacteria, was previously shown to exist in the extreme halophile, Halobacterium vallismortis. The results are consistent with the conclusion based upon 16S rRNA analyses that Methanomicrobiales and the extreme halophiles cluster together.Abbreviation DAHP 3-deoxy-d-arabino-heptulosonate-7-phosphate     

The biochemical basis for growth inhibition by L-phenylalanine in Neisseria gonorrhoeae

Clinical isolates of Neisseria gonorrhoeae are commonly subject to growth inhibition by phenylpyruvate or by L-phenylalanine. A blockade of tyrosine biosynthesis is indicated since inhibition is reversed by either L-tyrosine or 4-hydroxyphenylpyruvate. Phenylalanine-resistant (PheR) and phenylalanine-sensitive (PheS) isolates both have a single 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase that is partially inhibited by L-phenylalanine (80%). However, PheS and PheR isolates differ in that the ratio of phenylpyruvate aminotransferase to 4-hydroxyphenylpyruvate aminotransferase is distinctly greater in PheS isolates than in PheR isolates. A mechanism for growth inhibition is proposed in which phenylalanine exerts two interactive effects. (i) Phenylalanine decreases precursor flow to 4-hydroxyphenylpyruvate through its controlling effect upon DAHP synthase; and (ii) phenylalanine is largely transaminated to phenylpyruvate, which saturates both aminotransferases, preventing transamination of an already limited supply of 4-hydroxyphenylpyruvate to L-tyrosine.     

Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: quantification of metabolic impact

A quantitative analysis of the impact of feedback inhibition on aromatic amino acid biosynthesis was performed in chemostat cultures of Saccharomyces cerevisiae. Introduction of a tyrosine-insensitive allele of ARO4 (encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase) caused a three-fold increase of intracellular phenylalanine and tyrosine concentrations. These amino acids were not detected extracellularly. However, an over 100-fold increase of the extracellular levels of phenylacetate, phenylethanol and their para-hydroxyl analogues was observed. The total increase of the flux through the aromatic pathway was estimated to be over four-fold. Individual overexpression of either the wild-type or feedback insensitive allele of ARO7 (encoding chorismate mutase had no significant impact. However when they were combined with the Tyr-insensitive ARO4 allele in combination with the Tyr-insensitive ARO4 allele, extracellular concentrations of aromatic compounds were increased by over 200-fold relative to the reference strain, corresponding to a 4.5-fold increase of the flux through the aromatic amino acid biosynthesis pathway. Elimination of allosteric control on these two key reactions in aromatic amino acid metabolism significantly affected intracellular concentrations of several non-aromatic amino acids. This broader impact of amino acid biosynthesis presents a challenge in rational optimization of the production of specific amino acids and derived flavour compounds.