Anthranilate Synthase Gene Research Articles

Metabolic engineering of Corynebacterium glutamicum for l-tyrosine production from glucose and xylose

Microbial production of aromatic compounds is an attractive and sustainable biotechnological approach. With this motivation, here metabolic engineering of Corynebacterium glutamicum for l-tyrosine (l-Tyr) overproduction was attempted by pushing the carbon flux more towards l-Tyr. Translational start codon exchanges of prephenate dehydratase (pheA), anthranilate synthase (trpE), and phenylalanine aminotransferase (pat) genes revealed that reduced expression of pheA was the major contributor to increased l-Tyr titer while codon exchange in trpE was effective to a lower extent. Overexpression of aroE and qsuC, encoding shikimate dehydrogenase and 3-dehydroquinate dehydratase, respectively, and of dapC (cg1253), which is predicted to encode prephenate aminotransferase, were futile to increase l-Tyr titer. Similarly, deletion of the qsuABD gene cluster had also not enhanced titer. As for increasing precursor supply, deletion of ptsG of glucose uptake and overexpression of inositol permease (iolT2) and glucokinase (glcK) were not effective, but with utilization of xylose, enabled by overexpression of xylose isomerase (xylA) and xylulokinase (xylB), titer improved. Highest l-Tyr titer using the construct was 3.1 g/L on glucose and 3.6 g/L on a 1:3 (w/v) mixture of glucose and xylose. This result displays the potential of the constructed strain to produce l-Tyr from lignocellulosic renewable carbon sources.

Integration of the metabolome and transcriptome reveals indigo biosynthesis in Phaius flavus flowers under freezing treatment.

BackgroundIndigo-containing plant tissues change blue after a freezing treatment, which is accompanied by changes in indigo and its related compounds. Phaius flavus is one of the few monocot plants containing indigo. The change to blue after freezing was described to explore the biosynthesis of indigo in P. flavus.MethodsIn this study, we surveyed the dynamic change of P. flavus flower metabolomics and transcriptomics.ResultsThe non-targeted metabolomics and targeted metabolomics results revealed a total of 98 different metabolites, the contents of indole, indican, indigo, and indirubin were significantly different after the change to blue from the freezing treatment. A transcriptome analysis screened ten different genes related to indigo upstream biosynthesis, including three anthranilate synthase genes, two phosphoribosyl-anthranilate isomerase genes, one indole-3-glycerolphosphate synthase gene, five tryptophan synthase genes. In addition, we further candidate 37 cytochrome P450 enzyme genes, one uridine diphosphate glucosyltransferase gene, and 24 β-D-glucosidase genes were screened that may have participated in the downstream biosynthesis of indigo. This study explained the changes of indigo-related compounds at the metabolic level and gene expression level during the process of P. flavus under freezing and provided new insights for increasing the production of indigo-related compounds in P. flavus. In addition, transcriptome sequencing provides the basis for functional verification of the indigo biosynthesis key genes in P. flavus.

Sustainable Production of N-methylphenylalanine by Reductive Methylamination of Phenylpyruvate Using Engineered Corynebacterium glutamicum

N-alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for N-alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to N-alkylated amino acids like N-methyl-l-alanine or N-methylantranilate, a precursor of acridone alkaloids, have been established using engineered Corynebacterium glutamicum, which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of C. glutamicum for de novo production of N-methylphenylalanine based on reductive methylamination of phenylpyruvate. Pseudomonas putida Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes trpEG and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of l-tryptophan and l-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of DpkAP262A,M141L, N-methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an N-methylphenylalanine titer of 0.73 ± 0.05 g L−1, a volumetric productivity of 0.01 g L−1 h−1 and a yield of 0.052 g g−1 glucose were reached. When xylose isomerase gene xylA from Xanthomonas campestris and the endogenous xylulokinase gene xylB were expressed in addition, xylose as sole carbon source supported production of N-methylphenylalanine to a titer of 0.6 ± 0.04 g L−1 with a volumetric productivity of 0.008 g L−1 h−1 and a yield of 0.05 g g−1 xylose. Thus, a fermentative route to sustainable production of N-methylphenylalanine by recombinant C. glutamicum has been established.

Accumulation mechanism of indigo and indirubin in Polygonum tinctorium revealed by metabolite and transcriptome analysis

The harvest time of a plant usually affects the yield of its metabolites. Polygonum tinctorium (Polygonum tinctorium Ait., P. tinctorium) is an important cultivated plant that produces the natural blue dye indigo and the medicinal metabolite indirubin. To explore the mechanism of indigo and indirubin accumulation, the leaves of P. tinctorium at different harvest times, namely July, August, and October, were investigated by metabolite and de novo transcriptome analyses. The results revealed that the indigo and indirubin content varied at different harvest times. Moreover, their contents were significantly higher (p < 0.05) in August than in either July or October. Transcriptome analysis showed different gene expression patterns of P. tinctorium leaves in August. Almost all the identified differentially expressed genes (DEGs) that were responsible for the biosynthesis of indigo and indirubin, including four anthranilate synthase (AS) genes, two indole-3-glycerolphosphate synthase (IGPS) genes, three tryptophan synthase (TS) genes and seven cytochrome P450 (CYP450) genes were expressed at significantly higher levels in August (p < 0.05), which correlated with the increase of the two compounds. Overall, the results of this study provide useful information for understanding the accumulation mechanism of indigo and indirubin in P. tinctorium, which may promote the development of agricultural breeding and industrial production of P. tinctorium. Moreover, sequence data obtained by de novo transcriptome sequencing of P. tinctorium provides a platform for future molecular biological studies.

Physico-chemical characterization and transcriptome analysis of 5-methyltryptophan resistant lines in rice

Mutation breeding has brought significant contributions to the development of high value crops. It steered the first studies to generate plants with desired mutations of genes encoding key enzymes involved in important metabolic pathways. Molecular characterization of 5-methyl tryptophan (5-MT) resistant plants has revealed different base changes in alpha unit of anthranilate synthase (OsASA) gene that can lead to insensitivity to feedback inhibition of anthranilate synthase. The objective of this study was to perform in silico analysis of microarray data from five progressing time points during grain filling of rice. Results showed various differentially expressed genes. Enrichment of these genes revealed their roles in amino acid transportation during grain filling. Surprisingly, among all DEGs, only LOC_Os06g42560, a tryptophan synthase beta chain, was found to be directly related to tryptophan biosynthesis. It might affect amino acid content during grain filling. For physico-chemical analysis, different grain and eating qualities parameters were measured using mutant rice lines. Evaluation results showed that 5MT resistant-lines (5MT R-lines) showed approximately 60% chalkiness after milling although it had 20 times higher tryptophan content measured in μg/100 mg seeds. Taste quality of these 5MT R-lines in general was not affected significantly. However, other parameters such as peak time of viscosity and gelatinization temperature showed different results compared to the wildtype. Mutant lines generated in this study are important resources for high tryptophan content, although they have lower grain quality than the wildtype. They might be useful for developing new high nutrient rice varieties.

TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus

Small peptides formed from non-ribosomal peptide synthetases (NRPS) are bioactive molecules produced by many fungi including the genus Aspergillus. A subset of NRPS utilizes tryptophan and its precursor, the non-proteinogenic amino acid anthranilate, in synthesis of various metabolites such as Aspergillus fumigatus fumiquinazolines (Fqs) produced by the fmq gene cluster. The A. fumigatus genome contains two putative anthranilate synthases – a key enzyme in conversion of anthranilic acid to tryptophan – one beside the fmq cluster and one in a region of co-linearity with other Aspergillus spp. Only the gene found in the co-linear region, trpE, was involved in tryptophan biosynthesis. We found that site-specific mutations of the TrpE feedback domain resulted in significantly increased production of anthranilate, tryptophan, p-aminobenzoate and fumiquinazolines FqF and FqC. Supplementation with tryptophan restored metabolism to near wild type levels in the feedback mutants and suggested that synthesis of the tryptophan degradation product kynurenine could negatively impact Fq synthesis. The second putative anthranilate synthase gene next to the fmq cluster was termed icsA for its considerable identity to isochorismate synthases in bacteria. Although icsA had no impact on A. fumigatus Fq production, deletion and over-expression of icsA increased and decreased respectively aromatic amino acid levels suggesting that IcsA can draw from the cellular chorismate pool.

Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa.

Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA (Ser) gene) via a 56-bp core sequence within the attP/attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa. The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa.

Aniline Is an Inducer, and Not a Precursor, for Indole Derivatives in Rubrivivax benzoatilyticus JA2

Rubrivivax benzoatilyticus JA2 and other anoxygenic photosynthetic bacteria produce indole derivatives when exposed to aniline, a xenobiotic compound. Though this phenomenon has been reported previously, the role of aniline in the production of indoles is still a biochemical riddle. The present study aims at understanding the specific role of aniline (as precursor or stimulator) in the production of indoles and elucidating the biochemical pathway of indoles in aniline-exposed cells by using stable isotope approaches. Metabolic profiling revealed tryptophan accumulation only in aniline exposed cells along with indole 3-acetic acid (IAA) and indole 3-aldehyde (IAld), the two major catabolites of tryptophan. Deuterium labelled aniline feeding studies revealed that aniline is not a precursor of indoles in strain JA2. Further, production of indoles only in aniline-exposed cells suggests that aniline is an indoles stimulator. In addition, production of indoles depended on the presence of a carbon source, and production enhanced when carbon sources were added to the culture. Isotope labelled fumarate feeding identified, fumarate as the precursor of indole, indicating de novo synthesis of indoles. Glyphosate (shikimate pathway inhibitor) inhibited the indoles production, accumulation of tryptophan, IAA and IAld indicating that indoles synthesis in strain JA2 occurs via the de novo shikimate pathway. The up-regulation of anthranilate synthase gene and induction of anthranilate synthase activity correlated well with tryptophan production in strain JA2. Induction of tryptophan aminotransferase and tryptophan 2-monooxygenase activities corroborated well with IAA levels, suggesting that tryptophan catabolism occurs simultaneously in aniline exposed cells. Our study demonstrates that aniline (stress) stimulates tryptophan/indoles synthesis via the shikimate pathway by possibly modulating the metabolic pathway.

Changes in primary and secondary metabolite levels in response to gene targeting-mediated site-directed mutagenesis of the anthranilate synthase gene in rice.

Gene targeting (GT) via homologous recombination allows precise modification of a target gene of interest. In a previous study, we successfully used GT to produce rice plants accumulating high levels of free tryptophan (Trp) in mature seeds and young leaves via targeted modification of a gene encoding anthranilate synthase—a key enzyme of Trp biosynthesis. Here, we performed metabolome analysis in the leaves and mature seeds of GT plants. Of 72 metabolites detected in both organs, a total of 13, including Trp, involved in amino acid metabolism, accumulated to levels >1.5-fold higher than controls in both leaves and mature seeds of GT plants. Surprisingly, the contents of certain metabolites valuable for both humans and livestock, such as γ-aminobutyric acid and vitamin B, were significantly increased in mature seeds of GT plants. Moreover, untargeted analysis using LC-MS revealed that secondary metabolites, including an indole alkaloid, 2-[2-hydroxy-3-β-D-glucopyranosyloxy-1-(1H-indol-3-yl)propyl] tryptophan, also accumulate to higher levels in GT plants. Some of these metabolite changes in plants produced via GT are similar to those observed in plants over expressing mutated genes, thus demonstrating that in vivo protein engineering via GT can be an effective approach to metabolic engineering in crops.

Soybean transcription factor GmMYBZ2 represses catharanthine biosynthesis in hairy roots of Catharanthus roseus

Catharanthus roseus (L.) G. Don is a plant species known for its production of a variety of terpenoid indole alkaloids, many of which have pharmacological activities. Production of catharanthine in cell cultures or in hairy roots established by transformation with Agrobacterium rhizogenes is of interest because catharanthine can be chemically coupled to the abundant leaf alkaloid vindoline to form the valuable anticancer drug vinblastine. Here, we observed a high amount of catharanthine in hairy roots of C. roseus, established by infecting leaf explants with the A. rhizogenes >agropine-type A4 strain carrying plasmid pRi. T-DNA transfer from plasmid pRi into hairy roots was confirmed by PCR for the essential T-DNA genes rolA and rolB and the agropine synthesis gene ags. The results suggest that integration of T-DNA into the plant DNA plays a positive role on the catharanthine pathway in C. roseus hairy roots. Furthermore, co-transformation with the soybean transcription factor GmMYBZ2 indicated that GmMYBZ2 reduces the catharanthine production by alteration of expression of a number of genes linked to the pathway. Transcription levels of the zinc-finger transcription factor 1 gene ZCT1 were high, and the transcription levels of the anthranilate synthase gene ASα, the strictosidine synthase gene STR, and the key transcription factor gene octadecanoid-responsive Catharanthus APETALA2/ethylene response factor were low. In addition, GmMYBZ2 had a negative effect on the gene expression levels of A-type cyclin CYSA and B-type cyclin CYSB, which was correlated with a reduced growth rate of the hairy roots.

Tryptophan synthesis block-associated sugar response, a physiological marker for rapid selection ofArabidopsis thalianatransformants, marked with feedback-inhibition-insensitive anthranilate synthase gene

The feedback-insensitive anthranilate synthase (AS) gene was used as a selection marker for transformants of Arabidopsis thaliana. The mutant gene (mAS1-2) was constructed by substituting nucleotide at the effector-binding site of the intrinsic AS gene via PCR-mediated site-directed mutagenesis and flanked with the myrosinase promoter pyk10 to drive its expression during initial root elongation. This inducible gene cassette was first introduced into Agrobacterium tumefaciens and then delivered into A. thaliana by floral-dip inoculation. 5-methyltryptophan (5-MT) inhibited AS and suppressed seedling growth of wild type plants as a result of tryptophan starvation. With the addition of sucrose (10 mg/ml), 5-MT inhibited cotyledon opening and caused anthocyanin to accumulate in juvenile seedlings. The present mutant reversed the tryptophan starvation caused by 5-MT and blocked subsequent sugar responses. The sugar responses were detected in non-transformed plants grown on a selection medium containing 10 mg/ml of sucrose and 10 μg/ml of 5-MT after 3 days of incubation. Thus, true transformants could be selected after a short incubation, compared to the conventional kanamycin-selection method that did not eliminate all non-transformed plants.

In vitro studies indicate a quinone is involved in bacterial Mn(II) oxidation

Manganese(II)-oxidizing bacteria play an integral role in the cycling of Mn as well as other metals and organics. Prior work with Mn(II)-oxidizing bacteria suggested that Mn(II) oxidation involves a multicopper oxidase, but whether this enzyme directly catalyzes Mn(II) oxidation is unknown. For a clearer understanding of Mn(II) oxidation, we have undertaken biochemical studies in the model marine alpha-proteobacterium, Erythrobacter sp. strain SD21. The optimum pH for Mn(II)-oxidizing activity was 8.0 with a specific activity of 2.5 nmol x min(-1) x mg(-1) and a K (m) = 204 microM. The activity was soluble suggesting a cytoplasmic or periplasmic protein. Mn(III) was an intermediate in the oxidation of Mn(II) and likely the primary product of enzymatic oxidation. The activity was stimulated by pyrroloquinoline quinone (PQQ), NAD(+), and calcium but not by copper. In addition, PQQ rescued Pseudomonas putida MnB1 non Mn(II)-oxidizing mutants with insertions in the anthranilate synthase gene. The substrate and product of anthranilate synthase are intermediates in various quinone biosyntheses. Partially purified Mn(II) oxidase was enriched in quinones and had a UV/VIS absorption spectrum similar to a known quinone requiring enzyme but not to multicopper oxidases. These studies suggest that quinones may play an integral role in bacterial Mn(II) oxidation.

Expression of a feedback insensitive anthranilate synthase gene from tobacco increases free tryptophan in soybean plants

Soybean [Glycine max (L.) Merr.] embryogenic cultures were transformed by particle bombardment with the feedback-insensitive tobacco anthranilate synthase (AS) gene ASA2 driven by the CaMV 35S promoter and selected using hph as the selectable marker gene. Only one of eight regenerated lines that set seed and contained ASA2 expressed the gene highly and contained increased free tryptophan (Trp) levels in leaves, seeds and embryogenic cultures. Leaf extracts of the ASA2 expressing line contained about twice as much AS enzyme activity as the untransformed control and this activity was only slightly more feedback-insensitive. Amino acid analysis showed that both leaves and embryogenic tissue cultures of the ASA2 expressing line had four to five-times the normal levels of free Trp and slightly higher free tyrosine and phenylalanine. The seed total Trp content was only slightly increased. Metabolic profiling-analysis by GC-MS detected no other consistent differences. These studies show that the ASA2 gene can be expressed in soybean and that modest changes in Trp synthesis occurs.

Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation.

A total of 35 independent transgenic tobacco plants were produced using the Agrobacterium tumefaciens-leaf segment co-cultivation method followed by selection with kanamycin for the nptII gene. The vector also carried the tobacco feedback-insensitive anthranilate synthase gene (ASA2). Many of the lines showed increased ASA2 mRNA levels but only three contained increased free tryptophan (Trp) and many lines contained lower Trp than the untransformed control. The line with the highest Trp level (threefold that of the untransformed control) contained increased anthranilate synthase activity (AS) both in leaves and a cell suspension culture derived from the plant while the feedback insensitivity was most evident in the suspension culture. Other kinetic data also indicated that the ASA2 encoded AS alpha-subunit was more abundant in the tissue culture than in leaves. Progeny seedlings from this line were resistant to certain toxic Trp analogs, especially alpha-methyltryptophan (alphaMT) and less so to the most commonly used analog, 5-methyltryptophan. Shoots formed more readily from leaves of two of the transgenic lines than from leaves of the untransformed control on alphaMT, indicating that it might be possible to use ASA2 as a selectable marker gene and alphaMT as the selection agent.

Use of the tobacco feedback-insensitive anthranilate synthase gene (ASA2) as a selectable marker for legume hairy root transformation.

The feedback-insensitive anthranilate synthase ( ASA2) cDNA--isolated from a 5-methyltryptophan (5MT)-resistant tobacco cell line--driven by the CaMV 35S promoter or 606 bp of the native ASA2 promoter, was introduced into the forage legume plant Astragalus sinicus or soybean ( Glycine max), using Agrobacterium rhizogenes strains DC-AR2 or K599, respectively. Hairy roots of A. sinicus transformed with 35S-ASA2 but not 606- ASA2 could be directly selected using 20-75 micro M 5MT. ASA2 mRNA was expressed in all A. sinicus lines selected with 5MT, but nptII mRNA was expressed only in some lines even though the gene was present. Free tryptophan was increased 8- to 26-fold in A. sinicus and 3- to 6-fold in soybean (selected with kanamycin). An HPLC method was used to measure anthranilate synthase (AS) activity since there was a fluorescent compound or compounds present in the soybean hairy root extracts. The transformed soybean hairy roots contained more feedback-resistant AS activity, showing that there is interaction of the tobacco ASA2 alpha-subunit with the soybean beta-subunit to form an active enzyme. Soybean hairy roots that express ASA2 also exhibit 5MT resistance. These results demonstrate that the tobacco feedback-insensitive ASA2 gene can be used as a selectable marker for transformation of the legume A. sinicus.

Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine

Transgenic hairy roots of Catharanthus roseus were established with glucocorticoid inducible tryptophan decarboxylase (TDC) expression alone or in combination with inducible expression of a feedback-resistant anthranilate synthase alpha subunit (ASα) from Arabidopsis. Northern blot analysis confirmed transcription of the anthranilate synthase gene upon induction in the double line (TDC+ASα) and in vitro enzyme assays confirmed increased resistance to feedback inhibition by tryptophan. In TDC enzyme assays, increases of 48% and 87% in the TDC and double lines, respectively, were noted. Although the TDC line showed no significant increase in tryptamine levels on induction, induction of the double line resulted in increases in tryptamine levels of as much as six-fold for a 3 day late exponential induction. Downstream effects on alkaloids were noted in the TDC line where serpentine specific yields increased as much as 129% on induction. No effects on measured alkaloids were noted in the double line, but the two clones have very different basal alkaloid biosynthetic capacities. Within this study, the engineering of the indole pathway in C. roseus hairy roots is reported, and the role of the indole pathway in alkaloid biosynthesis explored.

A 5-methyltryptophan resistant mutant of rice has an altered regulation of anthranilate synthase gene expression

The rice mutant, TR-1, was previously selected for resistance to growth inhibition by the tryptophan (Trp) analog 5-methyltryptophan (5MT). This mutant has an elevated Trp level. Since anthranilate synthase (AS) catalyzes the first step of Trp biosynthesis and is normally subject to feedback regulation by Trp, alteration of AS causes elevated Trp levels. In this study, we performed a physiological, biochemical and molecular characterization of the 5MT resistant mutant TR-68, a derivative of TR-1. The extracts of TR-68 exhibited 6-fold higher AS activity than that of the wild-type under standard conditions. However, the AS activity of TR-68 was as sensitive to feedback inhibition by Trp as that of the wild-type. We determined the nucleotide sequences of a part of rice AS gene ( OASA1) corresponding to the site of mutation in 5MT resistant mutant in Arabidopsis and to the site of naturally feedback insensitive AS in Nicotiana tabacum and Ruta graveolens, however, there were no differences in these regions between TR-68 and wild-type. In the wild-type, the expression of two AS genes ( OASA1 and OASA2) was inhibited simultaneously by 5MT and the addition of Trp partly suppressed this inhibition. In contrast, 5MT did not affect the expression of AS genes in TR-68. These data indicate that the 5MT resistance of TR-68 may be caused by mutation of a regulator of the expression of the AS genes.

Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator.

The nucleotide sequence of a 2-4 kb BamHI-SalI fragment of Streptomyces venezuelae ISP5230 DNA that complements trpE and trpG mutations in Escherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two subunits of anthranilate synthase. The two-thirds nearest the amino terminus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit. Upstream of ORF2 is a small ORF encoding 18 amino acids that include three adjacent Trp residues; in addition the ORF contains inverted repeats with sequence and positional similarity to the products of attenuator (trpL) regions that regulate tryptophan biosynthesis in other bacteria. In cultures of a trpC mutant of S. venezuelae, increasing the concentration of exogenous tryptophan decreased the formation of anthranilate synthase; similar evidence of endproduct repression was obtained in a trpCER mutant of E. coli transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrations of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutation in E. coli. However, S. venezuelae transformants in which the two-base deletion had been introduced by replacement of homologous chromosomal DNA did not exhibit a Trp- phenotype. The result implies that S. venezuelae has one or more additional genes for anthranilate synthase. In alignments with anthranilate synthase genes from other organisms, ORF2 from S. venezuelae most closely resembled genes for phenazine biosynthesis in Pseudomonas. The results bear on the function of the gene in S. venezuelae.

The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): Genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae

The bacterial endosymbionts (Buchnera) from the aphids Rhopalosiphum padi, R. maidis, Schizaphis graminum, and Acyrthosiphon pisum contain the genes for anthranilate synthase (trpEG) on plasmids made up of one or more 3.6-kb units. Anthranilate synthase is the first as well as the rate-limiting enzyme in the tryptophan biosynthetic pathway. The amplification of trpEG on plasmids may result in an increase of enzyme protein and overproduction of this essential amino acid, which is required by the aphid host. The nucleotide sequence of trpEG from endosymbionts of different species of aphids is highly conserved, as is an approximately 500-bp upstream DNA segment which has the characteristics of an origin of replication. Phylogenetic analyses were performed using trpE and trpG from the endosymbionts of these four aphids as well as from the endosymbiont of Schlechtendalia chinensis, in which trpEG occurs on the chromosome. The resulting phylogeny was congruent with trees derived from sequences of two chromosome-located bacterial genes (part of trpB and 16S ribosomal DNA). In turn, trees obtained from plasmid-borne and bacterial chromosome-borne sequences were congruent with the tree resulting from phylogenetic analysis of three aphid mitochondrial regions (portions of the small and large ribosomal DNA subunits, as well as cytochrome oxidase II). Congruence of trees based on genes from host mitochondria and from bacteria adds to previous support for exclusively vertical transmission of the endosymbionts within aphid lineages. Congruence with trees based on plasmid-borne genes supports the origin of the plasmid-borne trpEG from the chromosomal genes of the same lineage and the absence of subsequent plasmid exchange among endosymbionts of different species of aphids.

The endosymbiont (Buchnera sp.) of the aphid Diuraphis noxia contains plasmids consisting of trpEG and tandem repeats of trpEG pseudogenes.

Most aphids are dependent for their survival on prokaryotic endosymbionts assigned to the genus Buchnera. Among the functions of Buchnera species is the synthesis of tryptophan, which is required by the aphid host. In Buchnera species from the aphid Diuraphis noxia, the genes for anthranilate synthase (trpEG) were found on a plasmid which consisted of seven tandem repeats of a 3.2-kb unit and one 2.6-kb unit which differed in containing a 0.6-kb deletion. One of the 3.2-kb units contained open reading frames corresponding to trpEG; the remaining units contained trpEG pseudogenes (psi). The nucleotide sequence upstream of trpE contained a region that has characteristics of an origin of replication (ori). Relative to trpB (a chromosomal gene), there were about two copies of the trpEG-containing plasmid. Comparisons of the nucleotide sequences of the 3.2-kb units containing trpEG and psi trpEG indicated that most changes occurred in a 700-nucleotide segment that included the region upstream of trpE and the portion of this gene coding for the N terminus. The consequence of these changes was the silencing of trpEG by inactivation of the putative promoter region and premature termination of the TrpE peptide. In contrast, the nucleotide sequence of the segment corresponding to ori was conserved in the units containing trpEG and psi trpEG. We offer a number of speculations on the evolutionary pressure in this lineage which resulted in the silencing of most of trpEG while still retaining the regions resembling ori.