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Kyo Wakasa - One of the best experts on this subject based on the ideXlab platform.
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integrated metabolomic and transcriptomic analyses of high tryptophan rice expressing a mutant Anthranilate Synthase alpha subunit
Journal of Experimental Botany, 2007Co-Authors: Joseph G Dubouzet, Atsushi Ishihara, Fumio Matsuda, Hisashi Miyagawa, Kyo Wakasa, Hiroyoshi IwataAbstract:Transgenic rice plants overexpressing a mutant rice gene for Anthranilate Synthase alpha subunit (OASA1D) accumulate large amounts of free tryptophan (Trp) with few adverse effects on the phenotype, except for poor germination and weak seedling growth. Metabolic profiling of 8-d-old seedlings of Nipponbare and two high-Trp lines, HW1 and HW5, by high performance liquid chromatography-photo diode array (HPLC-PDA) confirmed that, relative to Nipponbare, only the peak attributed to Trp was significantly changed in the profiles of the OASA1D lines. More detailed and targeted analysis using HPLC coupled with tandem mass spectrometry revealed that the OASA1D lines had higher levels of Anthranilate, tryptamine, and serotonin than Nipponbare, but these metabolites were at much lower levels than free Trp. The levels of phenylalanine (Phe) and tyrosine (Tyr) were not affected by the overproduction of Trp. Transcriptomic analysis by microarray validated by quantitative Real-Time PCR (qRT-PCR) revealed that at least 12 out of 21 500 genes showed significant differential expression among genotypes. Except for the OASA1D transgene and a putative IAA b-glucosyltransferase, these were not related to Trp metabolism. Most importantly, the overexpression of the OASA1D and the consequent accumulation of Trp in these lines had little effect on the overall transcriptome, consistent with the minimal effects on growth and the metabolome. Integrated analysis of the metabolome and transcriptome of these OASA1D transgenic lines indicates that the over-accumulation of free Trp may be partly due to the low activity of Trp decarboxylase or other metabolic genes that directly utilize Trp as a substrate.
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accumulation of free tryptophan in azuki bean vigna angularis induced by expression of a gene oasa1d for a modified α subunit of rice Anthranilate Synthase
Plant Science, 2006Co-Authors: Moemen S Hanafy, Shaikh M Rahman, Mutasim M Khalafalla, Hany A Elshemy, Yumi Nakamoto, Masao Ishimoto, Kyo WakasaAbstract:Abstract Anthranilate Synthase (AS) is a key metabolic enzyme of the tryptophan (Trp) biosynthetic pathway in plants and microbes, and its activity is regulated by feedback inhibition by Trp. A rice gene ( OASA1D ) for a modified α-subunit of Anthranilate Synthase that is resistant to such feedback inhibition was introduced into azuki bean [ Vigna angularis (Willd.) Ohwi & Ohashi] by Agrobacterium -mediated transformation followed by selection with hygromycin B. Five independent azuki bean lines expressing OASA1D in which the transgene was fixed in the T 2 generation were obtained. The free Trp level in seeds of these lines (1.3–3.3 mg/g of dry weight) was increased compared with that in wild-type seeds (0.2 mg/g). In contrast, the amount of protein-bound (fixed) Trp in seeds was not affected by expression of the transgene. The AS activity in the transgenic seeds showed a reduced sensitivity to feedback inhibition by Trp or the Trp analog 5-methyltryptophan, indicating that the rice OASA1D subunit was able to interact functionally with the β-subunit of azuki bean AS. These results reveal that transformation with OASA1D is a feasible approach to increase Trp synthesis and accumulation in mature seeds of grain legumes.
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metabolic changes in arabidopsis thaliana expressing the feedback resistant Anthranilate Synthase α subunit gene oasa1d
Phytochemistry, 2006Co-Authors: Atsushi Ishihara, Hisashi Miyagawa, Yohei Asada, Yoshitaka Takahashi, Naoto Yabe, Yoshibumi Komeda, Takaaki Nishioka, Kyo WakasaAbstract:Abstract Anthranilate Synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant Anthranilate Synthase α subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 μM l -Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.
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structure based in vitro engineering of the Anthranilate Synthase a metabolic key enzyme in the plant tryptophan pathway
Plant Physiology, 2005Co-Authors: Takuya Kanno, Kyo Wakasa, Akira Komatsu, Koji Kasai, Joseph G Dubouzet, Minako Sakurai, Yasuko Ikejirikanno, Yuzuru TozawaAbstract:Rice (Oryza sativa) Anthranilate Synthase α-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.
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characterization of tryptophan overproducing potato transgenic for a mutant rice Anthranilate Synthase α subunit gene oasa1d
Planta, 2005Co-Authors: Fumio Matsuda, Haruna Miyazawa, Hisashi Miyagawa, Tetsuya Yamada, Kyo WakasaAbstract:Potato plants (Solanum tuberosum cv. May Queen) transgenic for OASA1D, which encodes a point mutant of an α-subunit of rice (Oryza sativa) Anthranilate Synthase (AS, EC 4.1.3.27), were generated in order to determine the effects of the mutant gene on levels of free tryptophan (Trp) and AS activity in this important crop. Expression of OASA1D in potato induced a 2- to 20-fold increase in the amount of free Trp. This increase was likely due to a reduction in the sensitivity of AS containing the mutant α-subunit to feedback inhibition by Trp. Nontargeted metabolite profiling by high-performance liquid chromatography coupled with ultraviolet photodiode array detection as well as targeted profiling by liquid chromatography coupled with mass spectrometry revealed no marked changes in the levels of other metabolites, with the exception of indole-3-acetic acid (IAA), in the transgenic plants. The level of IAA in the upper part of the shoot was increased by a factor of 8.3–39, depending on the transgenic lines, with no detectable effect on plant growth or development. The effects of transformation thus appeared limited to the biosynthesis of Trp and IAA, with the overall metabolic network in potato being virtually unaffected. These results suggest that transformation with OASA1D may prove effective for the breeding of crops with an increased level of free Trp.
Ka-yiu San - One of the best experts on this subject based on the ideXlab platform.
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still stable after 11 years a catharanthus roseus hairy root line maintains inducible expression of Anthranilate Synthase
Biotechnology Progress, 2017Co-Authors: Jiayi Sun, Ka-yiu San, Christie A. M. PeeblesAbstract: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.
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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
Metabolic Engineering, 2011Co-Authors: Christie A. M. Peebles, Guy W. Sander, Jacqueline V. Shanks, Erik H Hughes, Ryan Peacock, Ka-yiu SanAbstract: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 horhammericine 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 horhammericine 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.
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five year maintenance of the inducible expression of Anthranilate Synthase in catharanthus roseus hairy roots
Biotechnology and Bioengineering, 2009Co-Authors: Christie A. M. Peebles, Guy W. Sander, Jacqueline V. Shanks, Ka-yiu SanAbstract: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.
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expression of the arabidopsis feedback insensitive Anthranilate Synthase holoenzyme and tryptophan decarboxylase genes in catharanthus roseus hairy roots
Journal of Biotechnology, 2006Co-Authors: Seung-beom Hong, Christie A. M. Peebles, Ka-yiu San, Jacqueline V. Shanks, Susan I. GibsonAbstract: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.
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Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of Anthranilate Synthase.
Biotechnology and bioengineering, 2006Co-Authors: Christie A. M. Peebles, Jacqueline V. Shanks, Susan I. Gibson, Seung-beom Hong, Ka-yiu SanAbstract: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 α 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 α or α and β 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 α subunit, we observed an increase of 125% in horhammericine 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 α and β 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. © 2005 Wiley Periodicals, inc.
Jack M Widholm - One of the best experts on this subject based on the ideXlab platform.
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co expression of the tobacco Anthranilate Synthase β subunit with its feedback insensitive α subunit as a selectable marker that also markedly increases the free tryptophan content
In Vitro Cellular & Developmental Biology – Plant, 2015Co-Authors: Xinghai Zhang, J. E. Brotherton, Jack M WidholmAbstract:Anthranilate Synthase (AS), a tetramer consisting of two α subunits and two β subunits, is the key control enzyme in the tryptophan (Trp) biosynthesis pathway. A naturally occurring α subunit of AS called ASA2 that is insensitive to Trp feedback inhibition was isolated from a tobacco suspension cell culture and has been extensively studied and used for both nuclear and plastid transformation. However, the obligate β subunit of AS had not been studied in tobacco. Therefore, the tobacco AS β subunit-encoding cDNA was cloned and its encoded protein was verified. Agrobacterium-mediated transformation of tobacco plants was performed, under the selection of the toxic Trp analogs, 7-methyltryptophan or α-methyltryptophan, with a construct containing both ASA2 and AS β subunit genes. Many transgenic plants overexpressing both subunits were identified and examined. Compared to the wild-type plants, the transgenic plants had higher levels of enzymatic activities for both holoenzyme and α subunit. The transgenic plants had 9 to 68 times the amount of free Trp as the wild-type plants, which was more pronounced than plants overexpressing ASA2 alone. This study demonstrates the potential of co-expressing AS α and β subunits as a robust plant transformation system as well as overcoming feedback inhibition to obtain high levels of Trp biosynthesis.
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tobacco plastid transformation using the feedback insensitive Anthranilate Synthase α subunit of tobacco asa2 as a new selectable marker
Journal of Experimental Botany, 2009Co-Authors: Pierluigi Barone, Xinghai Zhang, Jack M WidholmAbstract:Genetic engineering of chloroplasts normally requires the stable introduction of bacterial derived antibiotic or herbicide-resistance genes as selective markers. Ecological and health concerns have been raised due to the presence of such genes within the environment or the food supply. One way to overcome this issue is the use of plant genes able to confer a metabolic or developmental advantage to the transformed cells manipulating the plant's biosynthetic pathways. We explored the feasibility of using, for plastid transformation, the selection system based on the feedback-insensitive Anthranilate Synthase (AS) alpha-subunit gene of tobacco (ASA2) as a new selective marker and the indole analogue 4-methylindole (4MI) or the tryptophan analogue 7-methyl-DL-tryptophan (7MT) as the selection agents. An expression cassette containing Prrn-ASA2 was effectively integrated into the region between accD and ycf4 of the tobacco plastome by the biolistic process. Plastid transgenic plants were obtained on medium supplemented with 300 microM 7MT or 4MI. Transplastomic plants showed normal phenotype and fertility and the resistance to the selection agents 7MT and 4MI was transmitted maternally. The plastid transformed lines also exhibited a higher level of AS enzyme activity that was less sensitive to Trp-feedback inhibition and, consequently, increased free Trp levels in leaves about 7-fold.
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Use of 4-methylindole or 7-methyl-DL-tryptophan in a transformant selection system based on the feedback-insensitive Anthranilate Synthase α-subunit of tobacco (ASA2)
Plant Cell Reports, 2008Co-Authors: Pierluigi Barone, Jack M WidholmAbstract:Effective selectable markers are needed for basic research and commercial applications that do not involve antibiotic or herbicide resistance. A novel selection system based on a feedback-insensitive Anthranilate Synthase α-subunit of tobacco (ASA2) as selectable marker using either 4-methylindole (4MI) or 7-methyl- DL -tryptophan (7MT) as the selection agent was developed. We found that these two components were able to discriminate better between ASA2 expressing and untransformed lines than the most commonly used analog 5-methyltryptopan (5MT) in the seedling growth inhibition test. We successfully integrated an expression cassette containing an ASA2 cDNA driven by a cauliflower mosaic virus 35S promoter into tobacco leaf discs by A. tumefaciens and selected transgenic plants on medium supplemented with 300 μM of 7MT or 4MI. Due to the expression of the feedback-insensitive ASA2, the transgenic lines produced showed higher free tryptophan (Trp) concentrations than the untransformed WT control. These results demonstrate the feasibility of the selection system with the ASA2 gene in combination with the use of Trp or indole analogs as selective agent.
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expression of a feedback insensitive Anthranilate Synthase gene from tobacco increases free tryptophan in soybean plants
Plant Cell Reports, 2007Co-Authors: Yoshimi Inaba, J. E. Brotherton, Alexander V Ulanov, Jack M WidholmAbstract: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.
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use of the tobacco feedback insensitive Anthranilate Synthase gene asa2 as a selectable marker for legume hairy root transformation
Plant Cell Reports, 2004Co-Authors: Hyeonje Cho, J. E. Brotherton, Jack M WidholmAbstract: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 µM 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 α-subunit with the soybean β-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.
Christie A. M. Peebles - One of the best experts on this subject based on the ideXlab platform.
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still stable after 11 years a catharanthus roseus hairy root line maintains inducible expression of Anthranilate Synthase
Biotechnology Progress, 2017Co-Authors: Jiayi Sun, Ka-yiu San, Christie A. M. PeeblesAbstract: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.
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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
Metabolic Engineering, 2011Co-Authors: Christie A. M. Peebles, Guy W. Sander, Jacqueline V. Shanks, Erik H Hughes, Ryan Peacock, Ka-yiu SanAbstract: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 horhammericine 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 horhammericine 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.
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five year maintenance of the inducible expression of Anthranilate Synthase in catharanthus roseus hairy roots
Biotechnology and Bioengineering, 2009Co-Authors: Christie A. M. Peebles, Guy W. Sander, Jacqueline V. Shanks, Ka-yiu SanAbstract: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.
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expression of the arabidopsis feedback insensitive Anthranilate Synthase holoenzyme and tryptophan decarboxylase genes in catharanthus roseus hairy roots
Journal of Biotechnology, 2006Co-Authors: Seung-beom Hong, Christie A. M. Peebles, Ka-yiu San, Jacqueline V. Shanks, Susan I. GibsonAbstract: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.
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Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of Anthranilate Synthase.
Biotechnology and bioengineering, 2006Co-Authors: Christie A. M. Peebles, Jacqueline V. Shanks, Susan I. Gibson, Seung-beom Hong, Ka-yiu SanAbstract: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 α 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 α or α and β 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 α subunit, we observed an increase of 125% in horhammericine 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 α and β 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. © 2005 Wiley Periodicals, inc.
Yuzuru Tozawa - One of the best experts on this subject based on the ideXlab platform.
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structure based in vitro engineering of the Anthranilate Synthase a metabolic key enzyme in the plant tryptophan pathway
Plant Physiology, 2005Co-Authors: Takuya Kanno, Kyo Wakasa, Akira Komatsu, Koji Kasai, Joseph G Dubouzet, Minako Sakurai, Yasuko Ikejirikanno, Yuzuru TozawaAbstract:Rice (Oryza sativa) Anthranilate Synthase α-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.
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Use of a feedback-insensitive α subunit of Anthranilate Synthase as a selectable marker for transformation of rice and potato
Molecular Breeding, 2004Co-Authors: Tetsuya Yamada, Yuzuru Tozawa, Hisakazu Hasegawa, Teruhiko Terakawa, Yasunobu Ohkawa, Kyo WakasaAbstract:A selection system based on a mutant rice gene for a feedback-insensitive α subunit of Anthranilate Synthase ( OASA1D ) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.
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in vitro reconstitution of rice Anthranilate Synthase distinct functional properties of the alpha subunits oasa1 and oasa2
Plant Molecular Biology, 2004Co-Authors: Takuya Kanno, Kyo Wakasa, Yuzuru Tozawa, Koji Kasai, Yasuko IkejirikannoAbstract:Anthranilate Synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, α and β, and converts chorismate to Anthranilate. Two or more AS α-subunit genes have been identified and characterized in several land plants. Although α subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS α-subunit genes (OASA1 and OASA2) in rice (Oryza sativa). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS β subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two β subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a β subunit to achieve maximal enzymatic activity even with NH 4 + as the amino donor. The V max and K i for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.
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characterization of rice Anthranilate Synthase α subunit genesoasa1 and oasa2 tryptophan accumulation in transgenic rice expressing a feedback insensitive mutant of oasa1
Plant Physiology, 2001Co-Authors: Yuzuru Tozawa, Hisakazu Hasegawa, Teruhiko Terakawa, Kyo WakasaAbstract:Anthranilate Synthase (AS) is a key enzyme in the synthesis of tryptophan (Trp), indole-3-acetic acid, and indole alkaloids. Two genes, OASA1 and OASA2 , encoding AS α-subunits were isolated from a monocotyledonous plant, rice ( Oryza sativa cv Nipponbare), and were characterized. A phylogenetic tree of AS α-subunits from various species revealed a close evolutionary relationship among OASA1 and Arabidopsis ASA2, Ruta graveolens ASα2, and tobacco ASA2, whereas OASA2, Arabidopsis ASA1, and R. graveolens ASα1 were more distantly related. OASA1 is expressed in all tissues tested, but the amount of its mRNA was greater in panicles than in leaves and roots. The abundance of OASA2 transcripts is similar among tissues and greater than that of OASA1 transcripts; furthermore, OASA2 expression was induced by a chitin heptamer, a potent elicitor, suggesting that OASA2 participates in secondary metabolism. Expression of wild-type OASA1 or OASA2 transgenes did not affect the Trp content of rice calli or plants. However, transformed calli and plants expressing a mutated OASA1 gene, OASA1 (D323N), that encodes a protein in which aspartate-323 is replaced with asparagine manifested up to 180- and 35-fold increases, respectively, in Trp accumulation. These transgenic calli and plants were resistant to 300 μm 5-methyl-Trp, and AS activity of the calli showed a markedly reduced sensitivity to Trp. These results show that OASA1 is important in the regulation of free Trp concentration, and that mutation of OASA1 to render the encoded protein insensitive to feedback inhibition results in accumulation of Trp at high levels. The OASA1 (D323N) transgene may prove useful for the generation of crops with an increased Trp content.
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characterization of rice Anthranilate Synthase α subunit genes oasa1 and oasa2 tryptophan accumulation in transgenic rice expressing a feedback insensitive mutant of oasa1
Plant Physiology, 2001Co-Authors: Yuzuru Tozawa, Hisakazu Hasegawa, Teruhiko Terakawa, Kyo WakasaAbstract:Anthranilate Synthase (AS) is a key enzyme in the synthesis of tryptophan (Trp), indole-3-acetic acid, and indole alkaloids. Two genes, OASA1 and OASA2 , encoding AS α-subunits were isolated from a monocotyledonous plant, rice ( Oryza sativa cv Nipponbare), and were characterized. A phylogenetic tree of AS α-subunits from various species revealed a close evolutionary relationship among OASA1 and Arabidopsis ASA2, Ruta graveolens ASα2, and tobacco ASA2, whereas OASA2, Arabidopsis ASA1, and R. graveolens ASα1 were more distantly related. OASA1 is expressed in all tissues tested, but the amount of its mRNA was greater in panicles than in leaves and roots. The abundance of OASA2 transcripts is similar among tissues and greater than that of OASA1 transcripts; furthermore, OASA2 expression was induced by a chitin heptamer, a potent elicitor, suggesting that OASA2 participates in secondary metabolism. Expression of wild-type OASA1 or OASA2 transgenes did not affect the Trp content of rice calli or plants. However, transformed calli and plants expressing a mutated OASA1 gene, OASA1 (D323N), that encodes a protein in which aspartate-323 is replaced with asparagine manifested up to 180- and 35-fold increases, respectively, in Trp accumulation. These transgenic calli and plants were resistant to 300 μm 5-methyl-Trp, and AS activity of the calli showed a markedly reduced sensitivity to Trp. These results show that OASA1 is important in the regulation of free Trp concentration, and that mutation of OASA1 to render the encoded protein insensitive to feedback inhibition results in accumulation of Trp at high levels. The OASA1 (D323N) transgene may prove useful for the generation of crops with an increased Trp content.