• Journal of Microbiology and Biotechnology
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• v.29 no.6
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• pp.839-844
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• 2019

Anthranilate derivatives have been used as flavoring and fragrant agents for a long time. Recently, these compounds are gaining attention due to new biological functions including antinociceptive and analgesic activities. Three anthranilate derivatives, N-methylanthranilate, methyl anthranilate, and methyl N-methylanthranilate were synthesized using metabolically engineered stains of Escherichia coli. NMT encoding N-methyltransferase from Ruta graveolens, AMAT encoding anthraniloyl-coenzyme A (CoA):methanol acyltransferase from Vitis labrusca, and pqsA encoding anthranilate coenzyme A ligase from Pseudomonas aeruginosa were cloned and E. coli strains harboring these genes were used to synthesize the three desired compounds. E. coli mutants (metJ, trpD, tyrR mutants), which provide more anthranilate and/or S-adenosyl methionine, were used to increase the production of the synthesized compounds. MS/MS analysis was used to determine the structure of the products. Approximately, $185.3{\mu}M$ N-methylanthranilate and $95.2{\mu}M$ methyl N-methylanthranilate were synthesized. This is the first report about the synthesis of anthranilate derivatives in E. coli.

• YAKHAK HOEJI
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• v.40 no.4
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• pp.418-421
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• 1996

From the ethanol extract of the whole plant of Aconitum pseudo-laeve var. erectum Nakai, three new aromatic amides, methyl-N-acetyl anthranilate, methyl-N-(3-ethox ycarbonylpropionyl)anthranilate, methyl-N-(3-methoxycarbonylpropionyl) anthranilate were isolated and characterized on the basis of spectral data.

• KSBB Journal
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• v.10 no.1
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• pp.89-96
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• 1995

An Avabidofsis thaliana gene encoding phosphoribosyl anthranilate transferase is shown to be the gene that is defective in blue fluorescent trp 1 mutant plants. This gene, named PAT1, coding region is homologous to those for the phosphoribosyl anthranilate transferase from many microorganisms. This is due to a defect in tryptophan biosynthesis that leads to an accumulation of anthranilate, a fluorescent intermediate in the tryptophan pathway. PAT1 is a single-copy gene that complements all of the visible phenotypes of the different trp1 mutants. Experiments to determine the regulation of the PAT1 gene are in progress. The wild-type PAT1 promoter and several promoter deletions of PAT1 gene have been transformed into Arabidopsis tryptophan mutants. These constructs might identify promoter elements that control this patterns. We have isolated the homozygous lines in T3 seeds and analysed the protein levels using PAT antibody and PAT protein level increased two fold in pHSl07. Finally, the potential of using PAT1 as a selectable marker or visible reporter of gene expression is being explored.

• BMB Reports
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• v.32 no.1
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• pp.20-24
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• 1999

The first step of the branch pathway in tryptophan biosynthesis is catalyzed by anthranilate synthase, which is subjected to feedback inhibition by the end product of the pathway. The $trpE^{FBR}$ gene from a mutant Escherichia coli strain coding for anthranilate synthase that was insensitive to feedback inhibition by tryptophan has been cloned. To identify the amino acid changes involved in the feedback regulation of anthranilate synthase, the nucleotide sequence of the mutant $trpE^{FBR}$ gene was determined. Sequence analysis of the $trpE^{FBR}$ gene revealed that four bases were changed in the structural gene while alteration was not found in the 5' control region. Among these base changes, only two base substitutions caused the alterations in amino acid sequences. From the results of restriction fragment exchange mapping, the 61st nucleotide, C to A substitution, that changed $Pro^{21}{\rightarrow}Ser$ was identified as the cause of the desensitization to feedback inhibition by tryptophan. Additional feedback-resistant enzymes of the E. coli anthranilate synthases were constructed by site-directed mutagenesis to examine the effect of the $Ser^{40}\;{\rightarrow}\;Arg^{40}$ change found in the $trpE^{FBR}$ gene of Brevibacterium lactofermentum. From the feedback inhibition analysis, the $Pro^{21}{\rightarrow}Ser$ and $Ser^{40}{\rightarrow}Arg$ mutants maintained about 50% and 90% of their maximal activities, respectively, even at the extreme concentration of 10 mM tryptophan. From these results, we suggest that the $Pro^{21}$ and $Ser^{40}$ residues are involved in the tryptophan binding in the E. coli enzyme.

• Journal of Plant Biology
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• v.39 no.4
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• pp.243-247
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• 1996

Phosphoribosylanthranilate transferase (PAT) catalyzes the second step of the tryptophan biosynthetic pathway and is encoded by a single-copy gene that complements all the visible phenotypes of the tryptophan mutant (trp1-100) of Arabidopsis. The trp1-100 is blue fluorescent under UV light becuase it accumulates anthranilate. To obtain a plant with reduced PAT activity, PAT1 genes with several internal deletions in different promoter regions (pHS 101, pHS102, pHS104, pHS105, and pHS107) were induced into trp1-100 via Agrobacterium. Then, homozygous T3 plants were isolated and examined for blue fluorescence. Introduction of the PAT1 gene fusants results in the reversion of fluorescence phenotype except in the case of pHS105. These results prompted us to perform a parallel analysis of anthranilate synthase and PAT interms of the genetic complementation. A plant line carrying pHS105 gene fusant does not completely complement the blue fluorescence but it accumulates less anthranilate than trp1-100. The activity of PAT was reduced in the transgenic mutant as well. The plant carrying these constructs will add to the growing collection of molecular tools for the study of the indolic secondary metabolism.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.40 no.1
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• pp.52-58
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• 1995

5-methyltryptophan（5MT） resistant mutant plants （MRl） were analyzed for characterization of anthranilate synthetase （AS） and tryptophan synthetase （TS） enzymes. The enzyme was measured in crude extracts from MR1 and control seedlings of Danggin inbred line. There was no significant difference in the level of AS between MR1 and control seedlings when grown on MS medium without 5MT. However, MR1 seedlings grown on MS medium with 25mg/L 5MT showed the level of AS twice higher than that of control seedlings. The activity of AS was inhibited to 50％ in untreated plants when 4mg /L L-tryptophan was added to their extracts. Extracts from MR1 plants required about four times higher concentration of amino acid to cause equal inhibition. In the TS assay, the activity observed in MR1 seedlings was four times higher than that of control seedlings. We have also isolated and sequenced the gene which encoding the tryptophan synthetase B subunit （TSB） from maize. The gene encodes polypeptides with high homology to TSB isolated from other plants, and is expressed in all the developmental stages examined. Northern hybridization analysis indicated that the gene expression in MR1 seedlings grown on MS medium showed a higher level than in control seedlings.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1324-1327
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• 2015

The nucleotide sequence of the TRP1 gene encoding phosphoribosyl anthranilate isomerase in yeast Saccharomycopsis fibuligera was determined by degenerate polymerase chain reaction and genome walking. Sequence analysis revealed the presence of an uninterrupted open-reading frame of 759 bp, including the stop codon, encoding a 252 amino acid residue. The deduced amino acid sequence of Trp1 in S. fibuligera was 43.5% homologous to that of Komagataella pastoris. The cloned TRP1 gene (SfTRP1) complemented the trp1 mutation in Saccharomyces cerevisiae, suggesting that it encodes a functional TRP1 in S. fibuligera. A new auxotrophic marker to engineer starch-degrading yeast S. fibuligera is now available. The GenBank Accession No. for SfTRP1 is KR078268.

• Korean Journal of Pharmacognosy
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• v.20 no.4
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• pp.215-218
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• 1989

A new aromatic amide isolated from the hexane soluble fraction of the root of Aconitum pseudolaeve var. erectum has been characterized as methyl-N-(2-acetaminobenzoyl) anthranilate on the basis of spectroscopic data, and a monoglyceride was also isolated and identified as glycerol-1-hexadecanoate from the fraction.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1370-1375
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• 2023

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.186-195
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• 2015

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive ${\alpha}$ subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content.