Still stable after 11 years: A Catharanthus roseus Hairy root line maintains inducible expression of anthranilate synthase.

Hairy root cultures generated using Agrobacterium rhizogenes are an extensively investigated system for the overproduction of various secondary metabolite based pharmaceuticals and chemicals. This study demonstrated a transgenic Catharanthus roseus hairy root line carrying a feedback-insensitive anthranilate synthase (AS) maintained chemical and genetic stability for 11 years. The AS gene was originally inserted in the hairy root genome under the control of a glucocorticoid inducible promoter. After 11 years continuous maintenance of this hairy root line, genomic PCR of the ASA gene showed the presence of ASA gene in the genome. The mRNA level of AS was induced to 52-fold after feeding the inducer as compared to the uninduced control. The AS enzyme activity was 18.4 nmol/(min*mg) in the induced roots as compared to 2.1 nmol/(min*mg) in the control. In addition, the changes in terpenoid indole alkaloid concentrations after overexpressing AS were tracked over 11 years. The major alkaloid levels in induced and control roots at 11 years are comparable with the metabolite levels at 5 years. This study demonstrates the long term genetic and biochemical stability of hairy root lines, which has important implications for industrial scale applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:66-69, 2017.

Arabidopsis ERF1 Mediates Cross-Talk between Ethylene and Auxin Biosynthesis during Primary Root Elongation by Regulating ASA1 Expression.

The gaseous phytohormone ethylene participates in the regulation of root growth and development in Arabidopsis. It is known that root growth inhibition by ethylene involves auxin, which is partially mediated by the action of the WEAK ETHYLENE INSENSITIVE2/ANTHRANILATE SYNTHASE α1 (WEI2/ASA1), encoding a rate-limiting enzyme in tryptophan (Trp) biosynthesis, from which auxin is derived. However, the molecular mechanism by which ethylene decreases root growth via ASA1 is not understood. Here we report that the ethylene-responsive AP2 transcription factor, ETHYLENE RESPONSE FACTOR1 (ERF1), plays an important role in primary root elongation of Arabidopsis. Using loss- and gain-of-function transgenic lines as well as biochemical analysis, we demonstrate that ERF1 can directly up-regulate ASA1 by binding to its promoter, leading to auxin accumulation and ethylene-induced inhibition of root growth. This discloses one mechanism linking ethylene signaling and auxin biosynthesis in Arabidopsis roots.

The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots.

The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in tabersonine by 66% and hörhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in hörhammericine by 30%, lochnericine by 27% and tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

Five year maintenance of the inducible expression of anthranilate synthase in Catharanthus roseus hairy roots.

Transgenic hairy root cultures have the potential to be an industrial production platform for a variety of chemicals. This report demonstrates the long-term stability of a transgenic Catharanthus roseus hairy root line containing the inducible expression of a feedback-insensitive anthranilate synthase (AS). After 5 years in liquid culture, the presence of the inserted AS gene was confirmed by genomic PCR. The inducible expression of AS was confirmed by enzyme assay and by changes in terpenoid indole alkaloid concentrations. This report also demonstrates that it may take as long as 2 years for the metabolite profile to stabilize.

Characterization of two trpE genes encoding anthranilate synthase alpha-subunit in Azospirillum brasilense.

The previous report from our laboratory has recently identified a new trpE gene (termed trpE2) which exists independently in Azospirillum brasilense Yu62. In this study, amplification of trpE(G) (termed trpE1(G) here) confirmed that there are two copies of trpE gene, one trpE being fused into trpG while the other trpE existed independently. This is the first report to suggest that two copies of the trpE gene exist in this bacterium. Comparison of the nucleotide sequence demonstrated that putative leader peptide, terminator, and anti-terminator were found upstream of trpE1(G) while these sequence features did not exist in front of trpE2. The beta-galactosidase activity of an A. brasilense strain carrying a trpE2-lacZ fusion remained constant at different tryptophan concentrations, but the beta-galactosidase activity of the same strain carrying a trpE1(G)-lacZ fusion decreased as the tryptophan concentration increased. These data suggest that the expression of trpE1(G) is regulated at the transcriptional level by attenuation while trpE2 is constantly expressed. The anthranilate synthase assays with trpE1(G)- and trpE2- mutants demonstrated that TrpE1(G) fusion protein is feedback inhibited by tryptophan while TrpE2 protein is not. We also found that both trpE1(G) and trpE2 gene products were involved in IAA synthesis.

Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots.

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan, tryptamine, is a building block for the biosynthesis of terpenoid indole alkaloids. Previously, we manipulated the indole pathway by introducing an Arabidopsis feedback-insensitive anthranilate synthase (AS) alpha subunit (trp5) cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots [Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004a. Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol. Bioeng. 86, 718-727; Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004b. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6, 268-276]. Inducible expression of either or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In an attempt to more successfully engineer the indole pathway, a wild type Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic hairy roots expressing both trp5 and ASB1 show a significantly greater resistance to feedback inhibition of AS activity by tryptophan than plants expressing only trp5. In fact, a 4.5-fold higher concentration of tryptophan is required to achieve 50% inhibition of AS activity in plants overexpressing both genes than in plants expressing only trp5. In addition, upon a 3 day induction during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times more tryptophan (specific yield ca. 3.0 mg g(-1) dry weight) than the trp5 hairy root line. Concurrently, tryptamine levels increase up to 9-fold in the induced trp5:ASB1 line (specific yield ca. 1.9 mg g(-1) dry weight) as compared with only a 4-fold tryptamine increase in the induced trp5 line (specific yield ca. 0.3 mg g(-1) dry weight). However, endogenous TDC activities of both trp5:ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC hairy root line accumulates tryptamine up to 14-fold higher than the uninduced line. In parallel with the remarkable accumulation of tryptamine upon induction, alkaloid accumulation levels were significantly changed depending on the duration and dosage of induction.

Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase.

Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in hörhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.

Analysis of Spt7 function in the Saccharomyces cerevisiae SAGA coactivator complex.

The Saccharomyces cerevisiae SAGA complex is required for the normal transcription of a large number of genes. Complex integrity depends on three core subunits, Spt7, Spt20, and Ada1. We have investigated the role of Spt7 in the assembly and function of SAGA. Our results show that Spt7 is important in controlling the levels of the other core subunits and therefore of SAGA. In addition, partial SAGA complexes containing Spt7 can be assembled in the absence of both Spt20 and Ada1. Through biochemical and genetic analyses of a series of spt7 deletion mutants, we have identified a region of Spt7 required for interaction with the SAGA component Spt8. An adjacent Spt7 domain was found to be required for a processed form of Spt7 that is present in a previously identified altered form of SAGA called SLIK, SAGA(alt), or SALSA. Analysis of an spt7 mutant with greatly reduced levels of SLIK/SAGA(alt)/SALSA suggests a subtle role for this complex in transcription that may be redundant with a subset of SAGA functions.

Induction of anthranilate synthase activity by elicitors in oats.

Oat phytoalexins, avenanthramides, are a series of substituted hydroxycinnamic acid amides with anthranilate. The anthranilate in avenanthramides is biosynthesized by anthranilate synthase (AS, EC 4.1.3.27). Induction of anthranilate synthase activity was investigated in oat leaves treated with oligo-N-acetylchitooligosaccharide elicitors. AS activity increased transiently, peaking 6 h after the elicitation. The induction of activity was dependent on the concentration and the degree of polymerization of the oligo-N-acetylchitooligosaccharide elicitor. These findings indicate that the induction is part of a concerted biochemical change required for avenanthramide production. The elicitor-inducible AS activity was strongly inhibited by L-tryptophan and its analogues including 5-methyl-DL-tryptophan, and 5- and 6-fluoro-DL-tryptophan, while the activity was not affected by D-tryptophan. The accumulation of avenanthramide A was also inhibited by treatment of elicited leaves with these AS inhibitors, indicating that a feedback-sensitive AS is responsible for the avenanthramide production. In elicited leaves, the content of free anthranilate remained at a steady, low level during avenanthramide production. Moreover, administration of anthranilate to elicited oat leaves resulted in an enhanced avenanthramide accumulation. AS may play a role as a rate-limiting enzyme in the biosynthesis of avenanthramides.

Thermodynamics of reactions catalyzed by anthranilate synthase.

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq) + ammonia(aq) = anthranilate(aq) + pyruvate(aq) + H2O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq) + ammonia(aq) = ADIC(aq) + H2O(l) and ADIC(aq) = anthranilate(aq) + pyruvate(aq). The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively. Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79. Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate2-(aq) + NH4+(aq) = anthranilate-(aq) + pyruvate-(aq) + H+(aq) + H2O(l), delta rHmo = -(116.3 +/- 5.4) kJ mol-1. For the reaction: chorismate2-(aq) + NH4+(aq) = ADIC-(aq) + H2O(l), K = (20.3 +/- 4.5) and delta rHmo = (7.5 +/- 0.6) kJ mol-1. Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes delta rG'mo under approximately physiological conditions are given.

Expression of Sulfolobus solfataricus trpE and trpG genes in E. coli.

The genes trpE and trpG of the hyperthermophilic archaeon Sulfolobus solfataricus, encoding the components I and II of anthranilate synthase, were cloned and co-expressed in Escherichia coli. The properties of the recombinant protein were determined and compared to those of the wild type complex. Gel filtration chromatography revealed an alpha2beta2 composition. The heteromeric enzyme is fully active above 85 degrees C and can be considered to be an "extremozyme" according to Adams et al.[1]. Sulfolobus solfataricus anthranilate synthase is subject to feedback inhibition by L-tryptophan even if it lacks the co-operativity that has been observed for all the other tetrameric anthranilate synthases.

Immunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana.

In order to study the tryptophan biosynthetic enzymes of the plant Arabidopsis thaliana, polyclonal antibodies were raised against five of the tryptophan biosynthetic pathway proteins: anthranilate synthase alpha subunit, phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomerase, and the tryptophan synthase alpha and beta subunits. Immunoblot analysis of Arabidopsis leaf protein extracts revealed that the antibodies identify the corresponding proteins that are enriched in Arabidopsis chloroplast fractions. Precursors of phosphoribosylanthranilate isomerase and tryptophan synthase alpha subunit were synthesized by in vitro translation. The precursors were efficiently imported and processed by isolated spinach chloroplasts, and the cleavage sites within the precursors were determined. These results provide the first direct evidence that the tryptophan biosynthetic enzymes from Arabidopsis are synthesized as higher molecular weight precursors and then imported into chloroplasts and processed into their mature forms.

GCN4p activation of the yeast TRP3 gene is enhanced by ABF1p and uses a suboptimal TATA element.

Transcription of the TRP3 gene of Saccharomyces cerevisiae is regulated by GCN4p from a position proximal to the transcriptional initiation sites. The promoter's apparent lack of a conventional TATA element sequence has led it to be used as a model for TATA-less promoters. Through mutational analysis of the TRP3 promoter, we have identified two additional regulatory elements required for expression. The first, located 57 base pairs (bp) upstream of the GCN4p binding site, binds ABF1p in vitro. The ABF1p binding site was required for maximal levels of GCN4p-activated transcription in vivo; however, the -fold activation by GCN4p was not altered by ABF1p. The second element, positioned 23 bp downstream of the GCN4p binding site, contained the TATA-like sequence, TATTAA. This element was required for both basal and activated expression and almost certainly functions as a TATA-binding protein interaction site. Mutations that improved its TATA character for native or an altered specificity mutant of TATA-binding protein correspondingly improved its function. Interestingly, basal expression induced by ABF1p was virtually unchanged in the presence of point mutations in the TATTAA element. Furthermore, unlike the case for HIS3 where only a limited subset of TATA-like sequences can activate transcription in conjunction with GCN4p, many divergent TATA-like sequences allowed GCN4p activation of TRP3. We suggest that the apparent promoter specific use of these TATA elements by GCN4p results from ABF1p amplifying the GCN4p-induced expression to a detectable level.

Characterization of composite aminodeoxyisochorismate synthase and aminodeoxyisochorismate lyase activities of anthranilate synthase.

Anthranilate synthase [chorismate pyruvatelyase (amino-accepting), E.C.4.1.3.27] catalyzes the formation of anthranilate (o-aminobenzoate) and pyruvic acid from chorismate and glutamine. A mutant form of the enzyme from Salmonella typhimurium accumulates a compound that we had isolated and identified as trans-6-amino-5-[(1-carboxyethenyl)-oxy]-1,3- cyclohexadiene-1-carboxylic acid, commonly called aminodeoxyisochorismate (ADIC). Here we report that ADIC is formed by a reversible, Mg(2+)-dependent ADIC synthase activity of anthranilate synthase that can be functionally uncoupled from a Mg(2+)-dependent ADIC lyase activity of the enzyme by single amino acid substitutions in the TrpE subunit of the anthranilate synthase complex of S. typhimurium. Both of the component activities of the enzyme are sensitive to feedback inhibition by L-tryptophan. Purified ADIC is quantitatively converted to anthranilate and pyruvic acid by the ADIC lyase activity of wild-type anthranilate synthase. ADIC also serves as a substrate for the formation of chorismate by the enzyme in the absence of glutamine and (NH4)2SO4. The rate of ADIC formation by the mutant enzyme and the steady-state parameters for ADIC utilization by the wild-type enzyme are consistent with a role for ADIC as an enzyme-bound intermediate that does not accumulate during the course of the anthranilate synthase reaction. The altered catalytic specificity of mutant anthranilate synthase enzymes suggests a potential role for ADIC in secondary metabolism.

[Enhancing effect of chuangxinmycin on synthesis of enzymes in tryptophan synthesis pathway of chuangxinmycin-resistant mutant strain of E. coli].

Chuangxinmycin (CXM) and L-trp cause repression of trp-operon in wild strain of E. coli and indolepropanic acid (IPA) causes derepression of this strain. In CXM-resistant mutant strain, CXM and IPA both cause derepression, and interaction of CXM and IPA is competitive. However, L-trp results in neither repression nor derepression in the mutant, but it can counteract the activity of CXM or IPA. These results suggest that there are repression and derepression sites on repressor of E. coli. CXM can bind to both sites, but mainly to repression site. The structure of repression site of the CXM-resistant mutant is altered, so that CXM can not bind to the repression site and binds mainly to the derepression site, thus leading to the derepression of trp-operon.

Expression of the Escherichia coli trpE gene in E. coli K12 bacteria: maximum level, rate and time of initiation of anthranilate synthetase production.

We have investigated the effect of alterations in the structure of the plasmid-borne Escherichia coli tryptophan (trp) coding region and other regions of the same replicon on the level, rate and time of initiation of anthranilate synthetase component I (ASase) synthesis in E. coli K12. The maximum level of ASase produced corresponds to 60%-65% of the total cellular proteins. Adding sequences downstream of the trpE coding region decreases the level but does not affect the time of initiation and rate of trpE expression (ASase synthesis). The presence of additional protein coding sequences on the plasmid outside the trpE-A region causes ASase production to start earlier and decreases the rate of ASase synthesis. A second copy of the trpE coding sequences, if present within or outside the trpE-A coding region on the same replicon, doubles the rate of synthesis of ASase and slightly increases its final level of production. The initiation of ASase production occurs earlier when the two trpE copies are located within two distinct transcription units.

Complementation of a trpE deletion in Escherichia coli by Spirochaeta aurantia DNA encoding anthranilate synthetase component I activity.

A 2.7-kilobase Sau3A fragment of Spirochaeta aurantia DNA cloned in pBR322 complemented a trpE deletion in Escherichia coli. Deletion analysis and Tn5 mutagenesis of the resulting plasmid pBG100 defined a 2-kilobase-pair region that was required for both the complementation and the synthesis of 59,000- and 47,000-molecular-weight polypeptides (59K and 47K polypeptides) in maxicells. Both the 59K and the 47K polypeptides appear to be encoded by a single gene. A maxicell analysis of pBG100::Tn5 mutants suggests that the 47K polypeptide is not sufficient for the trpE complementation. In vitro and in vivo anthranilate synthetase (AS) assays indicate that the complementing activity encoded by pBG100 was functionally analogous to the AS component I of E. coli in that it utilized NH3 but not glutamine as the amino donor. pBG100 did not encode a glutamine amidotransferase activity, although the AS component I it encoded was capable of interacting with E. coli AS component II to catalyze the glutamine-requiring reaction. Expression appeared to depend on a promoter in the cloned S. aurantia DNA.

Studies on the involvement of the UGA readthrough process in the mechanism of attenuation of the tryptophan operon of Escherichia coli.

We asked if UGA suppression by charged tRNATrp, a process called UGA readthrough, is involved in the mechanism of attenuation of the tryptophan (trp) operon in Escherichia coli. For this purpose we used two mutations: strA(LD1) which causes restriction of UGA readthrough, and revA which partially overcomes the restriction of UGA readthrough caused by strA(LD1)(Engelberg-Kulka et al. 1982). trp attenuation was monitored by the regulation of the synthesis of the trp operon enzyme anthranilate synthetase (ASase) in trpR strains. We showed that the strA(LD1) mutation causes a significant increase in the level of synthesis of ASase in the presence of an excess of tryptophan, while the revA mutation reverses this effect, indicating that transcription termination at the trp attenuator site is relieved by restriction of UGA readthrough. Based on our results and the sequence data of the trp leader RNA of E. coli (Oxender et al. 1979), we offer a model for the involvement of the UGA readthrough process in trp attenuation. We suggest that the UGA readthrough process permits trp attenuation to respond to slight changes in the cellular concentration of charged tRNATrp.

Biochemical genetics of tryptophan synthesis in Pseudomonas acidovorans.

Sixty independent tryptophan auxotrophs of Pseudomonas acidovorans were isolated and characterized for nutritional response to intermediates of the pathway, accumulation of intermediates, and levels of tryptophan-synthetic enzymes. Mutants for each of the seven proteins catalyzing the five steps of tryptophan synthesis were obtained. Transductional analysis established three unlinked chromosomal regions: trpE, trpGDC, and trpFBA. The order of the genes within the two clusters was not determined. The levels and enzymatic activities of wild-type and mutant strains indicated that trpE and trpGDC were repressed by tryptophan. In contrast, trpFBA was not derepressed significantly by starvation for tryptophan. The trpG mutants had an additional requirement for p-aminobenzoate, which suggested that anthranilate synthase subunit II also served as glutamine-binding protein in the analogous reaction catalyzed by p-aminobenzoate synthase. In addition, trpD mutants revealed the ability of P. acidovorans to degrade anthranilate via the beta-ketoadipate pathway.