Engineering Escherichia coli membrane phospholipid head distribution improves tolerance and production of biorenewables.

Economically competitive microbial production of biorenewable fuels and chemicals is often impeded by toxicity of the product to the microbe. Membrane damage is often identified as a major mechanism of this toxicity. Prior efforts to strengthen the microbial membrane by changing the phospholipid distribution have largely focused on the fatty acid tails. Herein, a novel strategy of phospholipid head engineering is demonstrated in Escherichia coli. Specifically, increasing the expression of phosphatidylserine synthase (+pssA) was found to significantly increase both the tolerance and production of octanoic acid, a representative membrane-damaging solvent. Tolerance of other industrially-relevant inhibitors, such as furfural, acetate, toluene, ethanol and low pH was also increased. In addition to the increase in the relative abundance of the phosphoethanolamine (PE) head group in the +pssA strain, there were also changes in the fatty acid tail composition, resulting in an increase in average length, percent unsaturation and decreased abundance of cyclic rings. This +pssA strain had significant changes in: membrane integrity, surface potential, electrochemical potential and hydrophobicity; sensitivity to intracellular acidification; and distribution of the phospholipid tails, including an increase in average length and percent unsaturation and decreased abundance of cyclic rings. Molecular dynamics simulations demonstrated that the +PE membrane had increased resistance to penetration of ethanol into the hydrophobic core and also the membrane thickness. Further hybrid models in which only the head group distribution or fatty acid tail distribution was altered showed that the increase in PE content is responsible for the increase in bilayer thickness, but the increased hydrophobic core thickness is due to altered distribution of both the head groups and fatty acid tails. This work demonstrates the importance of consideration of the membrane head groups, as well as a modeling approach, in membrane engineering efforts.

Improving Escherichia coli membrane integrity and fatty acid production by expression tuning of FadL and OmpF.

BACKGROUND: Construction of microbial biocatalysts for the production of biorenewables at economically viable yields and titers is frequently hampered by product toxicity. Membrane damage is often deemed as the principal mechanism of this toxicity, particularly in regards to decreased membrane integrity. Previous studies have attempted to engineer the membrane with the goal of increasing membrane integrity. However, most of these works focused on engineering of phospholipids and efforts to identify membrane proteins that can be targeted to improve fatty acid production have been unsuccessful. RESULTS: Here we show that deletion of outer membrane protein ompF significantly increased membrane integrity, fatty acid tolerance and fatty acid production, possibly due to prevention of re-entry of short chain fatty acids. In contrast, deletion of fadL resulted in significantly decreased membrane integrity and fatty acid production. Consistently, increased expression of fadL remarkably increased membrane integrity and fatty acid tolerance while also increasing the final fatty acid titer. This 34% increase in the final fatty acid titer was possibly due to increased membrane lipid biosynthesis. Tuning of fadL expression showed that there is a positive relationship between fadL abundance and fatty acid production. Combinatorial deletion of ompF and increased expression of fadL were found to have an additive role in increasing membrane integrity, and was associated with a 53% increase the fatty acid titer, to 2.3 g/L. CONCLUSIONS: These results emphasize the importance of membrane proteins for maintaining membrane integrity and production of biorenewables, such as fatty acids, which expands the targets for membrane engineering.

Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables.

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.

Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity.

Carboxylic acids are an attractive biorenewable chemical, but as with many biorenewables, their toxicity to microbial biocatalysts limits their fermentative production. While it is generally accepted that membrane damage is the main mechanism of fatty acid toxicity, previous metabolic engineering efforts that increased membrane integrity did not enable increased carboxylic acid production. Here we used an evolutionary approach to improve tolerance to exogenous octanoic acid, with the goal of learning design strategies from this evolved strain. This evolution of an Escherichia coli MG1655 derivative at neutral pH in minimal media produced a strain with increased tolerance not only to octanoic acid, but also to hexanoic acid, decanoic acid, n-butanol and isobutanol. This evolved strain also produced carboxylic acids at a 5-fold higher titer than its parent strain when expressing the Anaerococcus tetradius thioesterase. While it has been previously suggested that intracellular acidification may contribute to carboxylic acid toxicity, we saw no evidence that the evolved strain has increased resistance to this acidification. Characterization of the evolved strain membrane showed that it had significantly altered membrane polarization (fluidity), integrity (leakage) and composition relative to its parent. The changes in membrane composition included a significant increase in average lipid length in a variety of growth conditions, including 30°C, 42°C, carboxylic acid challenge and ethanol challenge. The evolved strain has a more dynamic membrane composition, showing both a larger number of significant changes and larger fold changes in the relative abundance of membrane lipids. These results highlight the importance of the cell membrane in increasing microbial tolerance and production of biorenewable fuels and chemicals.

Metabolic flux analysis of Escherichia coli MG1655 under octanoic acid (C8) stress.

Systems metabolic engineering has made the renewable production of industrial chemicals a feasible alternative to modern operations. One major example of a renewable process is the production of carboxylic acids, such as octanoic acid (C8), from Escherichia coli, engineered to express thioesterase enzymes. C8, however, is toxic to E. coli above a certain concentration, which limits the final titer. (13)C metabolic flux analysis of E. coli was performed for both C8 stress and control conditions using NMR2Flux with isotopomer balancing. A mixture of labeled and unlabeled glucose was used as the sole carbon source for bacterial growth for (13)C flux analysis. By comparing the metabolic flux maps of the control condition and C8 stress condition, pathways that were altered under the stress condition were identified. C8 stress was found to reduce carbon flux in several pathways: the tricarboxylic acid (TCA) cycle, the CO2 production, and the pyruvate dehydrogenase pathway. Meanwhile, a few pathways became more active: the pyruvate oxidative pathway, and the extracellular acetate production. These results were statistically significant for three biological replicates between the control condition and C8 stress. As a working hypothesis, the following causes are proposed to be the main causes for growth inhibition and flux alteration for a cell under stress: membrane disruption, low activity of electron transport chain, and the activation of the pyruvate dehydrogenase regulator (PdhR).

Systems metabolic engineering design: fatty acid production as an emerging case study.

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C6 to C16 are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel-like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high-yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high-yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain-length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain-lengths and functionalities.

The ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid pathway.

BACKGROUND: The terpenoid indole alkaloid (TIA) pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. Unfortunately, these drugs are produced in trace amounts, causing them to be very costly. To increase production of these drugs, an improved understanding of the TIA regulatory pathway is needed. Towards this end, transgenic Catharanthus roseus hairy roots that overexpress the ORCA2 TIA transcriptional activator were generated and characterized. RESULTS: Transcriptional profiling experiments revealed that overexpression of ORCA2 results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the TIA pathway, and from the TIA pathway itself. In addition, metabolite-profiling experiments revealed that overexpression of ORCA2 significantly affects the levels of several TIA metabolites. ORCA2 overexpression also causes significant increases in transcript levels of several TIA regulators, including TIA transcriptional repressors. CONCLUSIONS: Results presented here indicate that ORCA2 plays a critical role in regulation of TIA metabolism. ORCA2 regulates expression of key genes from both feeder pathways, as well as the genes (STR and SGD) encoding the enzymes that catalyze the first two steps in TIA biosynthesis. ORCA2 may play an especially important role in regulation of the downstream branches of the TIA pathway, as it regulates four out of five genes characterized from this part of the pathway. Regulation of TIA transcriptional repressors by ORCA2 may provide a mechanism whereby increases in TIA metabolite levels in response to external stimuli are transient and limited in magnitude.

Influence of carbon to nitrogen ratios on soybean somatic embryo (cv. Jack) growth and composition.

Soybean [Glycine max (L.) Merr.] seed are valued for their protein and oil content. Soybean somatic embryos cultured in Soybean Histodifferentiation and Maturation (SHaM) medium were examined for their suitability as a model system for developing an understanding of assimilate partitioning and metabolic control points for protein and oil biosynthesis in soybean seed. This report describes the growth dynamics and compositional changes of SHaM embryos in response to change in the carbon to nitrogen ratio of the medium. It was postulated that at media compositions that were sufficient to support maximal growth rates, changes in the C:N ratio are likely to influence the partitioning of resources between the various storage products, especially protein and oil. As postulated, at steady-state growth rates, embryo protein content was strongly correlated with decreasing C:N ratios and increasing glutamine consumption rates. However, oil content remained relatively unchanged across the C:N ratio range tested, and resources were instead directed towards the starch and residual biomass (estimated by mass balance) pools in response to increasing C:N ratios. Protein and oil were inversely related only at concentrations of sucrose in the medium <88 mM, where carbon limited growth and no starch was found to accumulate in the tissues. These observations and the high reproducibility in the data indicate that SHaM embryos are an ideal model system for the application of metabolic flux analysis studies designed to test hypotheses regarding assimilate partitioning in developing soybean seeds.

Enhanced algal growth rate in a Taylor vortex reactor.

The rate of production of algal biomass in optically dense photobioreactors depends crucially on the temporal light exposure of microorganisms, which in turn is determined by fluid flow patterns and the quantity and spatial distribution of photosynthetically active radiation. In this report it is demonstrated that highly organized and robust toroidal flow structures known as Taylor vortices cause significant increases in the rate of biomass production, efficiency of light utilization, and CO2 uptake, and these effects become more pronounced at higher Reynolds numbers. In light of these findings and previously reported experiments using Taylor vortex flow to culture algae, it is argued that the flashing light effect, rather than mass transport effects, is responsible for the observed increases in the rate of photosynthesis.

An integrated computational and experimental study for overproducing fatty acids in Escherichia coli.

Increasing demands for petroleum have stimulated sustainable ways to produce chemicals and biofuels. Specifically, fatty acids of varying chain lengths (C6-C16) naturally synthesized in many organisms are promising starting points for the catalytic production of industrial chemicals and diesel-like biofuels. However, bio-production of fatty acids from plants and other microbial production hosts relies heavily on manipulating tightly regulated fatty acid biosynthetic pathways. In addition, precursors for fatty acids are used along other central metabolic pathways for the production of amino acids and biomass, which further complicates the engineering of microbial hosts for higher yields. Here, we demonstrate an iterative metabolic engineering effort that integrates computationally driven predictions and metabolic flux analysis techniques to meet this challenge. The OptForce procedure was used for suggesting and prioritizing genetic manipulations that overproduce fatty acids of different chain lengths from C6 to C16 starting with wild-type E. coli. We identified some common but mostly chain-specific genetic interventions alluding to the possibility of fine-tuning overproduction for specific fatty acid chain lengths. In accordance with the OptForce prioritization of interventions, fabZ and acyl-ACP thioesterase were upregulated and fadD was deleted to arrive at a strain that produces 1.70 g/L and 0.14 g fatty acid/g glucose (∼39% maximum theoretical yield) of C14₋16 fatty acids in minimal M9 medium. These results highlight the benefit of using computational strain design and flux analysis tools in the design of recombinant strains of E. coli to produce free fatty acids.

Metabolic analysis of wild-type Escherichia coli and a pyruvate dehydrogenase complex (PDHC)-deficient derivative reveals the role of PDHC in the fermentative metabolism of glucose.

Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of (13)C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional (13)C,(1)H NMR spectroscopy. Metabolite balancing, biosynthetic (13)C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO(2) and the calculated CO(2) evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO(2) for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO(2), thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO(2) and H(2)) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO(2) generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth.

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.

Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time.

Jasmonic acid (JA) activates the transcriptional regulator ORCA3, which has a role in regulating the terpenoid indole alkaloid (TIA) pathway within Catharanthus roseus. The TIA pathway leads to the production of the anticancer drugs vinblastine and vincristine. This work explores the transient effects of overexpressing ORCA3 under the control of a glucocorticoid-inducible promoter system in C. roseus hairy roots along with the simultaneous feeding of JA. The changes in TIA metabolites and in mRNA transcripts of pathway genes and regulators were tracked for 72h. Upon induction of ORCA3 expression and elicitation with JA, ORCA3 transcripts increased 170-fold whereas ORCA3 expression caused an 89-fold increase and JA elicitation caused a 5-fold increase in ORCA3 transcripts. JA treatment caused the largest increase in TIA metabolites and transcripts of pathway genes. These transcripts displayed a transient response with the maximum expression reached between 12 and 24h. In the samples overexpressing ORCA3, the largest increase in the transcripts of ZCT1 and ZCT2 (ZCT-zinc finger-binding protein), TIA transcriptional repressors, coincided with the largest increase in ORCA3 transcripts. This counter response of transcriptional repressors may explain why the large increase in ORCA3 transcripts do not correspond with larger increases in transcripts of TIA pathway genes.

The role of the octadecanoid pathway in the production of terpenoid indole alkaloids in Catharanthus roseus hairy roots under normal and UV-B stress conditions.

The octadecanoid pathway is responsible for producing jasmonic acid an important signaling molecule in plants, which controls the production of a variety of secondary metabolites. Previously the exogenous addition of jasmonic acid to Catharanthus roseus hairy roots caused an increase in terpenoid indole alkaloid (TIA) accumulation. The role of the endogenous production of jasmonic acid by the octadecanoid pathway in the production of TIAs in C. roseus hairy roots is examined. Feeding of octadecanoid pathway inhibitors suggests that the octadecanoid pathway does not actively control TIA production under normal growth conditions or during the UV-B stress response in C. roseus hairy roots.

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.

Metabolic flux maps comparing the effect of temperature on protein and oil biosynthesis in developing soybean cotyledons.

Metabolic flux maps developed from 13C metabolic flux analysis (13C MFA) are effective tools for assessing the response of biological systems to genetic or environmental perturbations, and for identifying possible metabolic engineering targets. Experimental treatments were designed to distinguish between temperature effects prior to, and during incubation in vitro, on primary metabolism in developing soybeans. Biomass accumulation increased with temperature as did carbon partitioning into lipids. The flux through the plastidic oxidative pentose phosphate pathway (pgl(P)) relative to sucrose intake remained fairly constant [ approximately 56% (+/-24%)] when cotyledons were transferred from an optimum growth temperature to varying temperatures in in vitro culture, signifying a rigid node under these conditions. However, pgl(P) flux ranged from 57 to 77% of sucrose intake when growth temperature in planta varied and were cultured in vitro at the same temperature (as the plant), indicating a flexible node for this case. The carbon flux through the anaplerotic reactions catalysed by plastidic malic enzyme (me(P)), cytosolic phosphoenolpyruvate (PEP) carboxylase and the malate (Mal) transporter from the cytosol to mitochondrion varied dramatically with temperature and had a direct influence on the carbon partitioning into protein and oil from the plastidic pyruvate (Pyr) pool. These results of the in vitro culture indicate that temperature during early stages of development has a dominant effect on establishing capacity for flux through certain components of central carbon metabolism.

Engineering of E. coli inherent fatty acid biosynthesis capacity to increase octanoic acid production.

BACKGROUND: As a versatile platform chemical, construction of microbial catalysts for free octanoic acid production from biorenewable feedstocks is a promising alternative to existing petroleum-based methods. However, the bio-production strategy has been restricted by the low capacity of E. coli inherent fatty acid biosynthesis. In this study, a combination of integrated computational and experimental approach was performed to manipulate the E. coli existing metabolic network, with the objective of improving bio-octanoic acid production. RESULTS: First, a customized OptForce methodology was run to predict a set of four genetic interventions required for production of octanoic acid at 90% of the theoretical yield. Subsequently, all the ten candidate proteins associated with the predicted interventions were regulated individually, as well as in contrast to the combination of interventions as suggested by the OptForce strategy. Among these enzymes, increased production of 3-hydroxy-acyl-ACP dehydratase (FabZ) resulted in the highest increase (+ 45%) in octanoic acid titer. But importantly, the combinatorial application of FabZ with the other interventions as suggested by OptForce further improved octanoic acid production, resulting in a high octanoic acid-producing E. coli strain +fabZ ΔfadE ΔfumAC ΔackA (TE10) (+ 61%). Optimization of TE10 expression, medium pH, and C:N ratio resulted in the identified strain producing 500 mg/L of C8 and 805 mg/L of total FAs, an 82 and 155% increase relative to wild-type MG1655 (TE10) in shake flasks. The best engineered strain produced with high selectivity (> 70%) and extracellularly (> 90%) up to 1 g/L free octanoic acid in minimal medium fed-batch culture. CONCLUSIONS: This work demonstrates the effectiveness of integration of computational strain design and experimental characterization as a starting point in rewiring metabolism for octanoic acid production. This result in conjunction with the results of other studies using OptForce in strain design demonstrates that this strategy may be also applicable to engineering E. coli for other customized bioproducts.

Flux quantification in central carbon metabolism of Catharanthus roseus hairy roots by 13C labeling and comprehensive bondomer balancing.

Methods for accurate and efficient quantification of metabolic fluxes are desirable in plant metabolic engineering and systems biology. Toward this objective, we introduce the application of "bondomers", a computationally efficient and intuitively appealing alternative to the commonly used isotopomer concept, to flux evaluation in plants, by using Catharanthus roseus hairy roots as a model system. We cultured the hairy roots on (5% w/w U-(13)C, 95% w/w naturally abundant) sucrose, and acquired two-dimensional [(13)C, (1)H] and [(1)H, (1)H] NMR spectra of hydrolyzed aqueous extract from the hairy roots. Analysis of these spectra yielded a data set of 116 bondomers of beta-glucans and proteinogenic amino acids from the hairy roots. Fluxes were evaluated from the bondomer data by using comprehensive bondomer balancing. We identified most fluxes in a three-compartmental model of central carbon metabolism with good precision. We observed parallel pentose phosphate pathways in the cytosol and the plastid with significantly different fluxes. The anaplerotic fluxes between phosphoenolpyruvate and oxaloacetate in the cytosol and between malate and pyruvate in the mitochondrion were relatively high (60.1+/-2.5 mol per 100 mol sucrose uptake, or 22.5+/-0.5 mol per 100 mol mitochondrial pyruvate dehydrogenase flux). The development of a comprehensive flux analysis tool for this plant hairy root system is expected to be valuable in assessing the metabolic impact of genetic or environmental changes, and this methodology can be extended to other plant systems.

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

Efforts to engineer Catharanthus roseus hairy roots to produce commercially significant amounts of valuable compounds, such as the terpenoid indole alkaloids vinblastine and vincristine, require the development of tools to study the effects of overexpressing key metabolic and regulatory genes. The use of inducible promoters allows researchers to control the timing and level of expression of genes of interest. In addition, use of inducible promoters allows researchers to use a single transgenic line as both the control and experimental line, minimizing the problems associated with clonal variation. We have previously characterized the use of a glucocorticoid-inducible promoter system to study the effects of gene overexpression within the terpenoid indole alkaloid pathway on metabolite production. Here the feasibility of using an ethanol-inducible promoter within C. roseus hairy roots is reported. This ethanol-inducible promoter is highly sensitive to ethanol concentration with a concentration of 0.005% ethanol causing a 6-fold increase in CAT reporter activity after 24 h of induction. The ethanol-inducible CAT activity increased 24-fold over a 72-h induction period with 0.5% ethanol.

Long-term maintenance of a transgenic Catharanthus roseus hairy root line.

Stably transformed transgenic hairy root cultures have the potential to be a valuable production platform for a variety of secondary metabolites. This study reports that a transgenic hairy root culture of Catharanthus roseus has been stably maintained for over 4.5 years. This culture carries a transgene that expresses the green fluorescent protein under the control of the glucocorticoid-inducible promoter. Genomic PCR confirmed the presence of the GFP insert within the hairy roots, and induction with dexamethasone caused a significant (p < 0.02) increase in GFP levels.