Biomedical subjects

Jacqueline V Shanks

Publications and source records attributed to Jacqueline V Shanks.

16 recordsLinked to original sources

PubMed2006

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.

Anthranilate Synthase↗

PubMed2006

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes.

We have established Catharanthus roseus hairy root cultures transgenic for the rol ABC genes from T(L)-DNA of the agropine-type Agrobacterium rhizogenes strain A4. The rol ABC hairy root lines exhibit a wild-type hairy root syndrome in terms of growth and morphology on solid medium. However, they differ from wild-type hairy root lines in that they more frequently have excellent adaptability to liquid medium and do not appear to form calli during cultivation. Moreover, they do not produce detectable levels of mannopine and agropine which, in contrast, are often synthesized abundantly in wild-type hairy root lines. The absence of these opines does not appear to cause the rol ABC lines to have higher levels of terpenoid indole alkaloids than wild-type hairy root lines. Unlike wild-type lines, rol ABC lines produce very similar levels of total alkaloids despite wide variations in individual alkaloid contents. This work demonstrates that the three genes rol ABC are sufficient to induce high-quality hairy roots in Catharanthus roseus.

Agrobacterium tumefaciens↗

PubMed2005

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.

Anthranilate Synthase↗

PubMed2004

Quantification of compartmented metabolic fluxes in developing soybean embryos by employing biosynthetically directed fractional (13)C labeling, two-dimensional [(13)C, (1)H] nuclear magnetic resonance, and comprehensive isotopomer balancing.

Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis tool that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-(13)C Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [(13)C, (1)H] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 (13)C isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain sds. A high flux was found through the oxidative pentose phosphate pathway (19.99 +/- 4.39 micromol d(-1) cotyledon(-1), or 104.2 carbon mol +/- 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 +/- 5.00 micromol d(-1) cotyledon(-1), or 113.2 carbon mol +/-26.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.

Carbon↗

PubMed2004

Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels.

Different plant species produce a variety of terpenoid indole alkaloids, which are of interest as plant defensive secondary metabolites and as valuable pharmaceuticals. Although significant progress has been made, the mechanisms regulating the levels of this important class of compounds require continued elucidation. Previous precursor feeding studies have indicated that alkaloid accumulation can be improved during the exponential growth phase of hairy root cultures through enhanced tryptophan availability. To test this relationship, transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of an Arabidopsis feedback-resistant anthranilate synthase alpha subunit. Enzyme assays demonstrated that the Arabidopsis alpha subunit is compatible with the native beta subunit and that anthranilate synthase activity is more resistant to tryptophan inhibition in induced than in uninduced extracts. The metabolic effects of expressing the feedback-resistant anthranilate synthase alpha subunit were also dramatic. Over a 6-day induction period during the late exponential growth phase, tryptophan and tryptamine specific yields increased from almost undetectable levels to 2.5 mg/g dry weight and from 25 microg/g to 267 microg/g dry weight, respectively. The greater than 300-fold increase in tryptophan levels observed in these studies under certain induction conditions compares favorably with the fold increases obtained in previous constitutive expression studies. Despite the large increases in tryptophan and tryptamine, the levels of most terpenoid indole alkaloids were not significantly altered, with the exception of lochnericine, which increased 81% after a 3-day induction period. These results suggest that terpenoid indole alkaloid levels are tightly controlled.

Anthranilate Synthase↗

PubMed2004

Improvements in metabolic flux analysis using carbon bond labeling experiments: bondomer balancing and Boolean function mapping.

The biosynthetically directed fractional (13)C labeling method for metabolic flux evaluation relies on performing a 2-D [(13)C, (1)H] NMR experiment on extracts from organisms cultured on a uniformly labeled carbon substrate. This article focuses on improvements in the interpretation of data obtained from such an experiment by employing the concept of bondomers. Bondomers take into account the natural abundance of (13)C; therefore many bondomers in a real network are zero, and can be precluded a priori--thus resulting in fewer balances. Using this method, we obtained a set of linear equations which can be solved to obtain analytical formulas for NMR-measurable quantities in terms of fluxes in glycolysis and the pentose phosphate pathways. For a specific case of this network with four degrees of freedom, a priori identifiability of the fluxes was shown possible for any set of fluxes. For a more general case with five degrees of freedom, the fluxes were shown identifiable for a representative set of fluxes. Minimal sets of measurements which best identify the fluxes are listed. Furthermore, we have delineated Boolean function mapping, a new method to iteratively simulate bondomer abundances or efficiently convert carbon skeleton rearrangement information to mapping matrices. The efficiency of this method is expected to be valuable while analyzing metabolic networks which are not completely known (such as in plant metabolism) or while implementing iterative bondomer balancing methods.

Algorithms↗

PubMed2004

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 (ASalpha) from Arabidopsis. Northern blot analysis confirmed transcription of the anthranilate synthase gene upon induction in the double line (TDC+ASalpha) 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.

Catharanthus↗

PubMed2002

Metabolic engineering of plants for alkaloid production.

Alkaloids purified from plants provide many pharmacologically active compounds, including leading chemotherapy drugs. As is generally true of secondary metabolites, overall productivity is low, making commercial production expensive. Alternative production methods remain impractical, leaving the plant as the best source for these valuable chemicals. Recently, significant progress in characterizing the biosynthetic pathways leading to various alkaloids has been made, and a number of relevant genes have been cloned. Metabolic engineering employing such genes provides a promising technology for improved productivity in plant cell cultures, plant tissue cultures, or intact plants. In exploring solutions though, metabolic engineers must be careful to recognize the limitations inherent in designing plant systems.

Alkaloids↗

PubMed2002

Quantification of metabolic flux in plant secondary metabolism by a biogenetic organizational approach.

Metabolic engineering represents a promising approach to enhance the yield of valuable natural products from plants. A method to quantify flux through metabolite measurements is necessary for the analysis of native and modified pathways. Rather than focusing only on the accumulation of the final products, analyzing a wide range of secondary metabolites has significant advantages. We propose a model that organizes the flux analysis by grouping metabolites of similar biosynthetic origin. To this end, we have quantified temporal profiles of metabolites from several branches of the indole alkaloid pathway in Catharanthus roseus hairy root cultures. By analyzing these data, we are able to examine the distribution of flux around key branchpoints. Furthermore, this analysis provides crucial information such as an estimate of total flux to secondary metabolism.

Aspidosperma↗

PubMedDate not supplied

Characterization of an inducible promoter system in Catharanthus roseus hairy roots.

Transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of green fluorescent protein (GFP), and GFP expression was characterized. The inducible system shows a tightly controlled, reversible, and dosage-dependent response to the glucocorticoid dexamethasone in C. roseus hairy roots. Full induction was noted after 12-18 h in the mature regions of the root tips and after 6 h in the meristem tissue. Upon removal of the inducing agent, GFP expression declined to undetectable levels in the mature tissues after 24 h and in the meristem after 48 h. Although no dosage-dependent response was noted in the meristem region, such a response was apparent in the mature region of the tip and verified by quantitative GFP analysis. The inducible promoter system allowed quantitative control of GFP expression between 0.01 and 10 microM dexamethasone with saturation occurring at higher levels. Using GFP as a model system allowed demonstration of the ability to control temporal and quantitative gene expression with the glucocorticoid-inducible promoter in transgenic C. roseus hairy roots.

Catharanthus↗

PubMedDate not supplied

Role of the non-mevalonate pathway in indole alkaloid production by Catharanthus roseus hairy roots.

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway) leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur in most bacteria and in all higher plants. Treatment with the antibiotic fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and lochnericine by Catharanthus roseus hairy root cultures in the exponential growth phase. However, fosmidomycin did not significantly affect alkaloid levels in stationary phase hairy root cultures. Feeding with 1-deoxy-D-xylulose, 10-hydroxygeraniol, or loganin resulted in significant increases in alkaloid production by exponential phase hairy root cultures. These results suggest that the DXP pathway is a major provider of carbon for the monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus hairy roots in the exponential phase.

Catharanthus↗

PubMedDate not supplied

Transient effects of overexpressing anthranilate synthase alpha and beta subunits in Catharanthus roseus hairy roots.

Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback-insensitive anthranilate synthase alpha subunit under the control of the glucocorticoid-inducible promoter system and the anthranilate synthase beta subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, hörhammericine, and tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h.

Anthranilate Synthase↗

PubMedDate not supplied

TNT phytotransformation pathway characteristics in Arabidopsis: role of aromatic hydroxylamines.

Basic knowledge of the plant transformation pathways of TNT will aid phytoremediation design and assessment. While TNT transformation by plant metabolism has been demonstrated in previous studies, the presence and role of hydroxylamines in the transformation pathway has not been sufficiently understood. Hydroxylamines are unequivocally shown to be formed by plant transformation of TNT by two axenic plant systems (Arabidopsis thaliana and Catharanthusroseus). In addition, confirmation was obtained for conversion of these hydroxylamines to previously identified conjugates. Further characteristics of TNT transformation in Arabidopsis, an increasingly popular model system for genetic and biochemical studies of TNT transformation, were elucidated by [U-14C]TNT mass balance studies and metabolite feeding studies. These studies showed the rapid conversion of TNT to unextractable bound compounds by Arabidopsis seedlings in agreement with the green-liver model. Arabidopsis seedlings formed and transformed 4-substituted metabolites more efficiently than the 2-substituted metabolites. A qualitative kinetic rate analysis of the pathway was performed to propose rate-limiting steps in the pathway and theoretical schemes for improved rates are suggested.

Arabidopsis↗

PubMedDate not supplied

Problem-based learning biotechnology courses in chemical engineering.

We have developed a series of upper undergraduate/graduate lecture and laboratory courses on biotechnological topics to supplement existing biochemical engineering, bioseparations, and biomedical engineering lecture courses. The laboratory courses are based on problem-based learning techniques, featuring two- and three-person teams, journaling, and performance rubrics for guidance and assessment. Participants initially have found them to be difficult, since they had little experience with problem-based learning. To increase enrollment, we are combining the laboratory courses into 2-credit groupings and allowing students to substitute one of them for the second of our 2-credit chemical engineering unit operations laboratory courses.

Biotechnology↗

PubMedDate not supplied

Phytotransformation of 2,4-dinitrotoluene in arabidopsis thaliana: toxicity, fate, and gene expression studies in vitro.

Basic knowledge of the plant transformation pathways and toxicity of 2,4-dinitrotoluene (2,4-DNT) will assist in the design and assessment of a phytoremediation strategy. This study presents the toxicity and fate of 2,4-DNT and gene expression in response to 2,4-DNT exposure using the model plant Arabidopsis thaliana, an increasingly popular system for genetic and biochemical studies of phytotransformation of explosives. From the results of biomass and root growth assays for toxicity, 2,4-DNT was toxic to the plants at concentrations as low as 1 mg/L. In the uptake study, 95% of the initial 2,4-DNT was removed by 15-day-old seedlings from liquid media regardless of the initial 2,4-DNT concentrations while 30% accounted for the adsorption to the autoclaved plant materials. The mass balance was over 86% using [U-14C]2,4-DNT, and the mineralization by the plants was less than 1% under sterile conditions during 14 days of exposure. The percentage of the bound radioactivity increased from 49% to 72% of the radioactivity in the plants, suggesting transformed products of 2,4-DNT may be incorporated into plant tissues such as lignin and cellulose. Monoaminonitrotoluene isomers and unknown metabolites with short retention times were detected as transformed products of 2,4-DNT by the plants. Most (68%) of the radioactivity taken up by the plants was in the root tissues in nonsterile hydroponic cultures. Glutathione and expression of related genes (GSH1 and GSH2) in plants exposed to 2,4-DNT were 1.7-fold increased compared to untreated plants. Genes of a glutathione S-transferase and a cytochrome P450, which were induced by 2,4,6-trinitrotoluene exposure in previous studies, were upregulated by 10- and 8-fold, respectively. The application of phytoremediation and the development of transgenic plants for 2,4-DNT may be based on TNT phytotransformation pathway characteristics because of the similar fate and gene expression in plants.

Arabidopsis↗

PubMedDate not supplied

Determination of biomass composition of Catharanthus roseus hairy roots for metabolic flux analysis.

Metabolic flux analysis is a powerful diagnostic tool in metabolic engineering, and determination of biomass composition is indispensable to accurate flux evaluation. We report the elemental and biomolecular composition of Catharanthus roseus hairy roots, a pharmaceutically significant plant system and an important metabolic engineering target. The molecular formula of the organic material in the hairy roots was C12.0H22.7N0.4O7.6 during mid-exponential growth. The abundances of lipids, lignin, cellulose, hemicellulose, starch, protein, proteinogenic amino acids, mineral ash, and moisture in the biomass were quantified. Analysis of water-soluble components of the biomass with 1-D 13C and 2-D [1H,1H] correlation (COSY) NMR spectroscopy revealed that the water-soluble components were composed almost entirely of -glucans. Agropine, a frequently reported hairy root biomass component, was not detected. Our measurements of the biomass components quantified 83.6 +/- 9.3% (w/w) of the biomass. Together with previously reported abundances of indole alkaloids, we accounted for at least 85.9 +/- 11.6% (w/w) of the carbon in the biomass, which enabled the precise determination of 12 biomass synthesis fluxes.

Biopolymers↗