Plant-based Production Systems Research Articles

Lignocellulosic Bioethanol Production: Perspectives and Challenges

Biofuels produced from different lignocellulosic materials have the potential to be a valuable substitute for gasoline. Many physicochemical structural and compositional factors hinder the hydrolysis of cellulose present in biomass to sugars and other organic compounds that can later be converted to fuels. Different pretreatment techniques can change the physical and chemical structure of the lignocellulosic biomass and improve their hydrolysis rates. Many methods have been shown to result in high sugar yields, above 90% of the theoretical yield. Advances in pretreatment and biological-processing steps clearly provide the greatest opportunity to reduce bioethanol costs, and much more emphasis is needed in these areas. For pretreatment, improved process configurations are needed that reduce chemical costs for hemicellulose hydrolysis and subsequent conditioning for biological processing. In addition, energy requirements for biomass milling and heating must be reduced, and less corrosive environments are desired to reduce the cost of vessels. Future biotechnology-based developments in processing technology will likely include: Improved cellulase and hemicellulase production economics via microbe or plant-based production systems; improved fermentation strains that efficiently utilize both hemicellulose and cellulosic sugars; consolidated bioprocessing microbes which combine the ability to break down cellulosic materials with the ability to efficiently ferment sugars to ethanol.

Towards development of a universal dengue vaccine - How close are we?

Dengue has been ranked as one of the top emerging diseases in Asia and Latin America. Current epidemiological data may not even reflect the true burden of disease due to under-reported figures. Vector control programmes have failed to contain the disease and worst of all, no specific treatment is available at the moment. Thereby, this pushes the demand for a dengue vaccine as a long-term protective approach. Despite there are numerous vaccine candidates ahead, they could be held back by different aspects in promoting vaccine implementation. Particularly for developing nations, logistics and cost are the major hurdles that need to be addressed in order to provide a quick yet affordable medical relief. As an alternative, plant-based vaccine production system is able to offer an attractive prospect given to its advantages of biocontainment warranty, low operation cost, rapid scalability and logistics flexibility. Researches that have embarked on this scope are laid out and reviewed in terms of the feasibility of plant system to serve as a biofactory for dengue vaccine.

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6% of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression.

Production of mono- and sesquiterpenes in Camelina sativa oilseed.

Camelina was bioengineered to accumulate (4 S )-limonene and (+)-δ-cadinene in seed. Plastidic localization of the recombinant enzymes resulted in higher yields than cytosolic localization. Overexpressing 1-deoxy- d -xylulose-5-phosphate synthase ( DXS ) further increased terpene accumulation. Many plant-derived compounds of high value for industrial or pharmaceutical applications originate from plant species that are not amenable to cultivation. Biotechnological production in low-input organisms is an attractive alternative. Several microbes are well established as biotechnological production platforms; however, their growth requires fermentation units, energy input, and nutrients. Plant-based production systems potentially allow the generation of high-value compounds on arable land with minimal input. Here we explore whether Camelina sativa (camelina), an emerging low-input non-foodstuff Brassicaceae oilseed crop grown on marginal lands or as a rotation crop on fallow land, can successfully be refactored to produce and store novel compounds in seed. As proof-of-concept, we use the cyclic monoterpene hydrocarbon (4S)-limonene and the bicyclic sesquiterpene hydrocarbon (+)-δ-cadinene, which have potential biofuel and industrial solvent applications. Post-translational translocation of the recombinant enzymes to the plastid with concurrent overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) resulted in the accumulation of (4S)-limonene and (+)-δ-cadinene up to 7mgg(-1) seed and 5mgg(-1) seed, respectively. This study presents the framework for rapid engineering of camelina oilseed production platforms for terpene-based high-value compounds.

Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

(+)-Valencene production in Nicotiana benthamiana is increased by down-regulation of competing pathways.

Plant sesquiterpenes, such as (+)-valencene, artemisinin, and farnesene are valuable chemicals for use as aromatics, pharmaceuticals, and biofuels. Plant-based production systems for terpenoids critically depend on the availability of farnesyl diphosphate (FPP). Currently, these systems show insufficient yields, due to the competition for FPP of newly introduced pathways with endogenous ones. In this study, for the first time an RNAi strategy aiming at silencing of endogenous pathways for increased (+)-valencene production was employed. Firstly, a transient production system for (+)-valencene in Nicotiana benthamiana was set up using agroinfiltration. Secondly, silencing of the endogenous 5-epi-aristolochene synthase (EAS) and squalene synthase (SQS) that compete for the FPP pool was deployed. This resulted in a N. benthamiana plant that produces (+)-valencene as a prevalent volatile with a 2.8-fold increased yield. Finally, the size of the FPP pool was increased by overexpression of enzymes that are rate-limiting in FPP biosynthesis. Combined with silencing of EAS and SQS, no further increase of (+)-valencene production was observed, but emission of farnesol. Formation of farnesol, which is a breakdown product of FPP, indicates that overproducing sesquiterpenes is no longer limited by FPP availability in the cytosol. This study shows that metabolic engineering of plants can effectively be used for increased production of desired products in plants.

Glyco-engineering for biopharmaceutical production in moss bioreactors.

The production of recombinant biopharmaceuticals (pharmaceutical proteins) is a strongly growing area in the pharmaceutical industry. While most products to date are produced in mammalian cell cultures, namely Chinese hamster ovary cells, plant-based production systems gained increasing acceptance over the last years. Different plant systems have been established which are suitable for standardization and precise control of cultivation conditions, thus meeting the criteria for pharmaceutical production. The majority of biopharmaceuticals comprise glycoproteins. Therefore, differences in protein glycosylation between humans and plants have to be taken into account and plant-specific glycosylation has to be eliminated to avoid adverse effects on quality, safety, and efficacy of the products. The basal land plant Physcomitrella patens (moss) has been employed for the recombinant production of high-value therapeutic target proteins (e.g., Vascular Endothelial Growth Factor, Complement Factor H, monoclonal antibodies, Erythropoietin). Being genetically excellently characterized and exceptionally amenable for precise gene targeting via homologous recombination, essential steps for the optimization of moss as a bioreactor for the production of recombinant proteins have been undertaken. Here, we discuss the glyco-engineering approaches to avoid non-human N- and O-glycosylation on target proteins produced in moss bioreactors.

Molecular pharming in plants and plant cell cultures: a great future ahead?

Plant biotechnology may not be a familiar concept to the general public, but it is a rapidly developing field of research that involves the use of plants, plant tissues and plant cell cultures to make or modify products and processes. The versatility of plants and plant cells can be harnessed to produce diverse products, including valuable proteins. This is often described as ‘molecular farming’ and it requires the introduction of foreign DNA into plants or plant cells, turning them into factories for the production of specific recombinant protein products. The term ‘molecular pharming’ is often used instead to highlight the production of protein-based biopharmaceuticals, which contributes to the sustainable production of drugs that promote human and animal wellbeing. Both terms also apply to the production of valuable secondary metabolites such as the anticancer drugs paclitaxel, vincristine and vinblastine, but we will focus on recombinant proteins and their use as biopharmaceuticals in this article. The biopharmaceutical markets have expanded rapidly over the last 20 years, and are projected to more than double in volume over the next decade from US$200 billion in 2013 to at least US$500 billion in 2020. The two major biopharmaceutical production systems are microbes (mainly Escherichia coli and yeast) and mammalian cells such as the Chinese hamster ovary platform. In both cases, productivity has increased substantially over the last decade due to process optimization, platform standardization and genetic improvements. Both the US FDA and European Medicines Agency are familiar with these systems, and standard protocols can be followed to ensure the approval of new products. However, equivalent protocols are only just emerging for plant-based production systems, and only one plant-derived biopharmaceutical protein is currently on the market. With their established production infrastructure and regulatory framework, microbial and mammalian production systems have raced far ahead of their plant-based counterparts. No company will change their production host without a clear economic benefit, nor will they consider plants and plant cells for new products if there is no advantage over their incumbent technology. Furthermore, new companies will not base their manufacturing on a second-best option. Therefore, plant-based systems must begin to compete head-to-head with the established systems and, on a technological basis, we can already identify the areas where plantbased systems have the advantage, namely in terms of speed, improved product quality and scalability. The international success story of molecular pharming began in 2006 with the US Department of Agriculture approval of a poultry vaccine against Newcastle disease developed by Dow AgroSciences (IN, USA) [1,2]. The vaccine was manufactured in transgenic tobacco cell suspension cultures and was a benchmark for the regulatory acceptance of plants as a manufacturing platform, Molecular pharming in plants and plant cell cultures: a great future ahead?

Production, characterization, and antigen specificity of recombinant 62‐71‐3, a candidate monoclonal antibody for rabies prophylaxis in humans

Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed in Nicotiana benthamiana. Glycosylation was analyzed by mass spectroscopy; functionality was confirmed by antigen ELISA, as well as rabies and pseudotype virus neutralization. Epitope characterization was performed using pseudotype virus expressing mutagenized rabies glycoproteins. Purified mAb demonstrated potent viral neutralization at 500 IU/mg. A critical role for antigenic site I of the glycoprotein, as well as for two specific amino acid residues (K226 and G229) within site I, was identified with regard to mAb 62-71-3 neutralization. Pseudotype viruses expressing glycoprotein from lyssaviruses known not to be neutralized by this antibody were the controls. The results provide the molecular rationale for developing 62-71-3 mAb for rabies PEP; they also establish the basis for developing an inexpensive plant-based antibody product to benefit low-income families in developing countries.—Both, L., van Dolleweerd, C., Wright, E., Banyard, A. C., Bulmer-Thomas, B., Selden, D., Altmann, F., Fooks, A. R., Ma, J. K.-C. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans.

Recombinant antibody production in Arabidopsis seeds triggers an unfolded protein response.

Among the many plant-based production systems that are being tested for molecular farming, seeds are very attractive, as they provide a stable environment in which the accumulating recombinant proteins can be stored. However, it is not known exactly how high production levels of recombinant antibodies influence the endogenous transcriptome and proteome of the developing seed. To address this question, we studied the transcriptomic status in developing Arabidopsis (Arabidopsis thaliana) seeds 13 d post anthesis of three transgenic lines, producing varying levels of recombinant VHH or single-chain Fv antibody fragments fused to the human immunoglobulin G1-derived Fc fragment under the control of the β-PHASEOLIN seed-specific promoter. Using genome-wide Tiling arrays, we demonstrated that only a small proportion of the transcriptome was significantly changed in each of the lines compared with the wild type. Strikingly, in all three lines, we found a large overlap of up-regulated genes corresponding to protein folding, glycosylation/modification, translocation, vesicle transport, and protein degradation, suggestive of a state of cellular stress called the unfolded protein response. Moreover, the gene up-regulation amplitude was similar in all three lines. We hypothesize that the production of recombinant antibodies in the endoplasmic reticulum triggers endoplasmic reticulum stress, causing a disturbance of the normal cellular homeostasis.

Whole Farm Net Greenhouse Gas Abatement from Establishing Kikuyu-Based Perennial Pastures in South-Western Australia.

Simple SummaryGreenhouse gas (GHG) emissions from ruminant livestock production (sheep, cattle and goats) have contributed to a common perception that a shift in the human diet from animal to plant-based products is environmentally responsible. In this study we found that the level of net emissions from livestock production systems is strongly influenced by the type of farming system that is used, and in fact GHG emission levels from some livestock production systems may be comparable with cropping systems. By introducing into farming systems ‘perennial’ pasture plants that are able to capture more atmospheric carbon, which is then stored in the soil, emission levels from livestock production can be substantially reduced.On-farm activities that reduce GHG emissions or sequester carbon from the atmosphere to compensate for anthropogenic emissions are currently being evaluated by the Australian Government as carbon offset opportunities. The aim of this study was to examine the implications of establishing and grazing Kikuyu pastures, integrated as part of a mixed Merino sheep and cropping system, as a carbon offset mechanism. For the assessment of changes in net greenhouse gas emissions, results from a combination of whole farm economic and livestock models were used (MIDAS and GrassGro). Net GHG emissions were determined by deducting increased emissions from introducing this practice change (increased methane and nitrous oxide emissions due to higher stocking rates) from the soil carbon sequestered from growing the Kikuyu pasture. Our results indicate that livestock systems using perennial pastures may have substantially lower net GHG emissions, and reduced GHG intensity of production, compared with annual plant-based production systems. Soil carbon accumulation by converting 45% of arable land within a farm enterprise to Kikuyu-based pasture was determined to be 0.80 t CO2-e farm ha−1 yr−1 and increased GHG emissions (leakage) was 0.19 t CO2-e farm ha−1 yr−1. The net benefit of this practice change was 0.61 t CO2-e farm ha−1 yr−1 while the rate of soil carbon accumulation remains constant. The use of perennial pastures improved the efficiency of animal production almost eight fold when expressed as carbon dioxide equivalent emissions per unit of animal product. The strategy of using perennial pasture to improve production levels and store additional carbon in the soil demonstrates how livestock should be considered in farming systems as both sources and sinks for GHG abatement.

Expression of biologically active human insulin‐like growth factor 1 in Arabidopsis thaliana seeds via oleosin fusion technology

Novel protein expression in plant-based systems has become an important tool in producing and studying therapeutic proteins. Among many plant-based systems developed so far, oleosin fusion technology is one of the most cost-effective and convenient methods. In this study, an important therapeutic protein, human insulin-like growth factor 1 (hIGF-1), was expressed in Arabidopsis thaliana seeds via this technology. The plant bias codon usage-optimized hIGF-1 gene was fused to the C-terminal of A. thaliana 18.5 kDa oleosin gene, and the fusion gene driven by an oleosin promoter was transferred into A. thaliana ecotype Col-0. The accumulation of oleosin-hIGF-1 fusion protein in transgenic seeds was up to 0.75% of total seed protein (TSP) and the expression level of hIGF-1 was 0.17% of the TSP, which was eight times higher than previously reported using other plant-based hIGF-1 production systems. The biological activity of the hIGF-1 as an oleosin-hIGF-1 fusion protein in vitro was demonstrated by using human SH-SY5Y neuroblastoma cells.

Assessing energy efficiencies and greenhouse gas emissions under bioethanol-oriented paddy rice production in northern Japan

To establish energetically and environmentally viable paddy rice-based bioethanol production systems in northern Japan, it is important to implement appropriately selected agronomic practice options during the rice cultivation step. In this context, effects of rice variety (conventional vs. high-yielding) and rice straw management (return to vs. removal from the paddy field) on energy inputs from fuels and consumption of materials, greenhouse gas emissions (fuel and material consumption-derived CO2 emissions as well as paddy soil CH4 and N2O emissions) and ethanol yields were assessed. The estimated ethanol yield from the high-yielding rice variety, “Kita-aoba” was 2.94 kL ha−1, a 32% increase from the conventional rice variety, “Kirara 397”. Under conventional rice production in northern Japan (conventional rice variety and straw returned to the paddy), raising seedlings, mechanical field operations, transportation of harvested unhulled brown rice and consumption of materials (seeds, fertilizers, biocides and agricultural machinery) amounted to 28.5 GJ ha−1 in energy inputs. The total energy input was increased by 14% by using the high-yielding variety and straw removal, owing to increased requirements for fuels in harvesting and transporting harvested rice as well as in collecting, loading and transporting rice straw. In terms of energy efficiency, the variation among rice variety and straw management scenarios regarding rice varieties and rice straw management was small (28.5–32.6 GJ ha−1 or 10.1–14.0 MJ L−1). Meanwhile, CO2-equivalent greenhouse gas emissions varied considerably from scenario to scenario, as straw management had significant impacts on CH4 emissions from paddy soils. When rice straw was incorporated into the soil, total CO2-equivalent greenhouse gas emissions for “Kirara 397” and “Kita-aoba” were 25.5 and 28.2 Mg CO2 ha−1, respectively; however, these emissions were reduced notably for the two varieties when rice straw was removed from the paddy fields in an effort to mitigate CH4 emissions. Thus, rice straw removal avers itself a key practice with respect to lessening the impacts of greenhouse gas emissions in paddy rice-based ethanol production systems in northern Japan. More crucially, the rice straw removed is available for ethanol production and generation of heat energy with a biomass boiler, all elements required for biomass-to-ethanol transformation steps including saccharification, fermentation and distillation. This indicates opportunities for further improvement in energy efficiency and reductions in greenhouse gas emissions under whole rice plant-based bioethanol production systems.

의약용 당단백질에 부가된 당사슬의 중요성

High value-added therapeutic proteins have been leading the biologics industry and occupied major portion of the market. More than 60% of the currently available protein therapeutics are glycoproteins attached with glycans which play crucial roles for the protein folding, therapeutic efficacy, in vivo half-life and immunogenecity. This review introduces the process of glycosylation and the impacts of glycans in the aspects of therapeutics. The important glycan structures in therapeutic performances were also summarized focusing on three representative categories of glycoproteins, cytokines, therapeutic antibody and enzyme. Currently, mammalian expression systems such as Chinese hamster ovary cells are preferred for the production of therapeutic glycoproteins due to their ability to synthesize glycans having similar structures with human type glycans. However, recent advances of plant glycoengineering to overcome the limitation originating from different glycan structures will soon allow to develop more efficient and economic plant-based production systems for therapeutic glycoproteins.

The abundance of nitrogen cycle genes amoA and nifH depends on land-uses and soil types in South-Eastern Australia

Abstract Nitrogen is a critical nutrient in plant-based primary production systems, therefore measurements of N cycling by microorganisms may add value to agricultural soil monitoring programs. Bacterial-mediated nitrogen cycling was investigated in soils from two broad land-uses (managed and remnant vegetation) across different Soil Orders from three geomorphic zones in Victoria, Australia, by examining the abundance of the genes amoA and nifH using quantitative polymerase chain reaction (qPCR). The aim of the study was to identify parameters influencing bacterial populations possessing the genes nifH and amoA, and examine their distribution at a regional scale across different management treatments. The gene amoA was most abundant in the neutral to slightly alkaline surface soils from Calcarosols in North-West Victoria. There was a highly significant (P

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

Characterization of green‐tissue protein extract from alfalfa (Medicago sativa) exploiting a 3‐D technique

There is a growing interest of pharmaceutical companies for plant-based production systems. To facilitate the general acceptance of plants as bioreactors, the establishment of efficient downstream operations is critical. It has been proposed that a better understanding of the properties of the contaminant proteins can benefit downstream processing design and operation. The coupled application of 2-DE with aqueous two-phase partitioning has been suggested as a practical 3-D method to characterize potential contaminant proteins from plant extracts. The application of this novel 3-D approach to a complex protein extract from alfalfa (Medicago sativa) containing a model recombinant protein (human granulocyte colony stimulating factor (hG-CSF)) resulted in the quantification of 55 protein spots. The 3-D properties (M(r), pI, and K(p)) obtained for 17 proteins comprising 69% of the alfalfa proteins, allowed the proposal of a prefractionation step as well as the identification of the target molecule (rG-CSF) from bulk of alfalfa proteins. The information obtained from this experimental approach was useful for the identification of the potential contaminant proteins that will occur in alfalfa when this plant is used as a host for recombinant proteins. Additionally, this method will assist in the design of adequate purification strategies for recombinant proteins expressed in alfalfa green tissue.

Progress in kalata peptide production via plant cell bioprocessing

Cyclotides are disulfide-rich mini-proteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. They typically comprise 28-37 amino acids and are produced from linear precursors, and translational modification via oxidative folding, proteolytic processing and N-C cyclization. Because these plant-derived peptides are resistant to degradation and do exhibit a diverse range of biological activities, they have become important agronomic and industrial objectives. Due to its tolerance to sequence variation, the cyclotide backbone is also potentially useful as a molecular scaffold for protein-engineering applications. Several production options are available for bioactive plant metabolites including natural harvesting, total chemical synthesis, and expression of plant pathways in microbial systems. For the cyclotides with low yields in nature, chemical complexity and lack of knowledge of the complete biosynthetic pathway, however, many of these options are precluded. Plant cell-culture technology shows promise towards the goal of producing therapeutically active cyclotides in quality and quantities required for drug development as they are amenable to process optimization, scale-up, and metabolic engineering. It is conceivable that plant-based production systems may ultimately prove to be the preferred route for the production of native or designed cyclotides, and will contribute towards the development of target-specific drugs.

Methods for Growing Nonfood Products in Transgenic Plants

The relatively high cost of producing select industrial and pharmaceutical products in traditional hosts has led many groups to investigate the benefits of plant-based production systems. Transgenic plants can offer significant cost advantages for select products, but the advantage can be eroded occasionally by the cost of confinement to segregate them from commodity crops. The conceptual similarities and differences between transgenic plant production systems and other transgenic hosts are discussed in regard to regulations and public perception. A system of regulated transgenic production is described that is based on dedicating an area solely to industrial products. This system would take advantage of surrounding crops that are also grown for industrial applications to offset the cost of growing the regulated transgenic plants. Examples are given that demonstrate the economic advantages of the system while concurrently creating a clearer distinction between food and nonfood products. This system may also increase public confidence, as it is modeled on current systems used to produce regulated transgenic products in nonplant hosts.

Engineered chloroplasts as vaccine factories to combat bioterrorism

Bacillus anthracis is ideal for making biological weapons, but the licensed anthrax vaccine is unsuitable for widespread public administration. Recombinant subunit-vaccine candidates offer potential alternatives, and plant-based production systems facilitate the inexpensive bulking of target antigens. A recent report demonstrates expression of anthrax protective antigen in tobacco chloroplasts--this material is immunogenic and protective when injected into mice. Provided an economic purification scheme can be developed, this technology holds promise for an improved vaccine.