[Show abstract][Hide abstract] ABSTRACT: The apocarotenoid crocetin and its glycosylated derivatives, crocins, confer the red colour to saffron. Crocetin biosynthesis in saffron is catalysed by the carotenoid cleavage dioxygenase CCD2 (AIG94929). No homologues have been identified in other plant species due to the very limited presence of crocetin and its derivatives in the plant kingdom.Spring Crocus species with yellow flowers accumulate crocins in the stigma and tepals. Four carotenoid CCDs, namely CaCCD1, CaCCD2 and CaCCD4a/b and CaCCD4c were first cloned and characterized.CaCCD2 was localized in plastids, and a longer CCD2 version, CsCCD2L, was also localized in this compartment. The activity of CaCCD2 was assessed in Escherichia coli and in a stable rice gene function characterization system, demonstrating the production of crocetin in both systems. The expression of all isolated CCDs was evaluated in stigma and tepals at three key developmental stages in relation with apocarotenoid accumulation. CaCCD2 expression parallels crocin accumulation, but C14 apocarotenoids most likely are associated to the CaCCD1 activity in Crocus ancyrensis flowers.The specific CCD2 localization and its membrane interaction will contribute to the development of a better understanding of the mechanism of crocetin biosynthesis and regulation in the chromoplast.
New Phytologist 09/2015; DOI:10.1111/nph.13609 · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins.
[Show abstract][Hide abstract] ABSTRACT: Molecular pharming is a cost-effective platform for the production of recombinant proteins in plants. Although the biopharmaceutical industry still relies on a small number of standardized fermentation-based technologies for the production of recombinant proteins there is now a greater awareness of the advantages of molecular pharming particularly in niche markets. Here we discuss some of the technical, economic and regulatory barriers that constrain the clinical development and commercialization of plant-derived pharmaceutical proteins. We also discuss strategies to increase productivity and product quality/homogeneity. The advantages of whole plants should be welcomed by the industry because this will help to reduce the cost of goods and therefore expand the biopharmaceutical market into untapped sectors.
[Show abstract][Hide abstract] ABSTRACT: Carotenoids are nutritionally-beneficial organic tetraterpenoid pigments synthesized mainly by plants, bacteria and fungi. Although research has focused on the production of carotenoids in staple crops to improve nutritional welfare in developing countries, there is also an enormous market for carotenoids in the industrialized world, where they are produced both as commodities and luxury goods targeted at the pharmaceutical, nutraceutical, food/feed additive, cosmetics and fine chemicals sectors. Carotenoids are economically valuable because they have diverse bioactive and chemical properties. Some are essential nutrients (e.g. β-carotene), others are antioxidants with specific roles (e.g. lutein and zeaxanthin) or general health-promoting roles that reduce the risk or progression of diseases associated with oxidative stress (e.g. lycopene), and still others are natural pigments (e.g. astaxanthin, which is added to fish feed to impart a desirable pink flesh color). Even carotenoid degradation products, such as damascones and damascenones, are used as fragrances in the perfumes industry. Here we discuss the importance of carotenoids in different market sectors, review current methods for commercial production and its regulation, summarize the most relevant patents and consider evidence supporting the health claims made by different industry sectors, focusing on case studies representing the most commercially valuable carotenoids on the market: β-carotene, lycopene, lutein, zeaxanthin and astaxanthin.
[Show abstract][Hide abstract] ABSTRACT: The population of sub-Saharan Africa is at risk from multiple, poverty-related endemic diseases. HIV and malaria are the most prevalent, but they disproportionately affect different groups of people, i.e. HIV predominantly affects sexually-active adults whereas malaria has a greater impact on children and pregnant women. Nevertheless, there is a significant geographical and epidemiological overlap which results in bidirectional and synergistic interactions with important consequences for public health. The immunosuppressive effects of HIV increase the risk of infection when individuals are exposed to malaria parasites and also the severity of malaria symptoms. Similarly, acute malaria can induce a temporary increase in the HIV viral load. HIV is associated with a wide range of opportunistic infections that can be misdiagnosed as malaria, resulting in the wasteful misuse of antimalarial drugs and a failure to address the genuine cause of the disease. There is also a cumulative risk of toxicity when antiretroviral and antimalarial drugs are given to the same patients. Synergistic approaches involving the control of malaria as a strategy to fight HIV/AIDS and vice versa are therefore needed in co-endemic areas. Plant biotechnology has emerged as a promising approach to tackle poverty-related diseases because plant-derived drugs and vaccines can be produced inexpensively in developing countries and may be distributed using agricultural infrastructure without the need for a cold chain. Here we explore some of the potential contributions of plant biotechnology and its integration into broader multidisciplinary public health programs to combat the two diseases in developing countries.
[Show abstract][Hide abstract] ABSTRACT: Metabolic engineering can be used to modulate endogenous metabolic pathways in plants or introduce new metabolic capabilities in order to increase the production of a desirable compound or reduce the accumulation of an undesirable one. In practice, there are several major challenges that need to be overcome, such as gaining enough knowledge about the endogenous pathways to understand the best intervention points, identifying and sourcing the most suitable metabolic genes, expressing those genes in such a way as to produce a functional enzyme in a heterologous background, and, finally, achieving the accumulation of target compounds without harming the host plant. This article discusses the strategies that have been developed to engineer complex metabolic pathways in plants, focusing on recent technological developments that allow the most significant bottlenecks to be overcome. Expected final online publication date for the Annual Review of Plant Biology Volume 65 is April 29, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
[Show abstract][Hide abstract] ABSTRACT: The do-it-yourself patent search is a useful alternative to professional patent analysis particularly in the context of publicly funded projects where funds for IP activities may be limited. As a case study, we analysed patents related to the engineering of terpenoid indole alkaloid (TIA) metabolism in plants. We developed a focused search strategy to remove redundancy and reduce the workload without missing important and relevant patents. This resulted in the identification of approximately 50 key patents associated with TIA metabolic engineering in plants, which could form the basis of a more detailed freedom-to-operate analysis. The structural elements of this search strategy could easily be transferred to other contexts, making it a useful generic model for publicly funded research projects.
[Show abstract][Hide abstract] ABSTRACT: Rice endosperm is devoid of carotenoids because the initial biosynthetic steps are absent. The early carotenogenesis reactions were constituted through co-transformation of endosperm-derived rice callus with phytoene synthase and phytoene desaturase transgenes. Subsequent steps in the pathway such as cyclization and hydroxylation reactions were catalyzed by endogenous rice enzymes in the endosperm. The carotenoid pathway was extended further by including a bacterial ketolase gene able to form astaxanthin, a high value carotenoid which is not a typical plant carotenoid. In addition to astaxanthin and precursors, a carotenoid accumulated in the transgenic callus which did not fit into the pathway to astaxanthin. This was subsequently identified as 4-keto-α-carotene by HPLC co-chromatography, chemical modification, mass spectrometry and the reconstruction of its biosynthesis pathway in Escherichia coli. We postulate that this keto carotenoid is formed from α-carotene which accumulates by combined reactions of the heterologous gene products and endogenous rice endosperm cyclization reactions.
[Show abstract][Hide abstract] ABSTRACT: The food production and processing value chain is under pressure from all sides—increasing demand driven by a growing and more affluent population; dwindling resources caused by urbanization, land erosion, pollution and competing agriculture such as biofuels; and increasing constraints on production methods driven by consumers and regulators demanding higher quality, reduced chemical use, and most of all environmentally beneficial practices ‘from farm to fork’. This pressure can only be addressed by developing efficient and sustainable agricultural practices that are harmonized throughout the value chain, so that renewable resources can be exploited without damaging the environment. Bridges must, therefore, be built between the diverse areas within the food production and processing value chain, including bridges between different stages of production, between currently unlinked agronomic practices, and between the different levels and areas of research to achieve joined-up thinking within the industry, so that the wider impact of different technologies, practices and materials on productivity and sustainability is understood at the local, regional, national and global scales. In this article, we consider the challenges at different stages and levels of the value chain and how new technologies and strategies could be used to build bridges and achieve more sustainable food/feed production in the future.
[Show abstract][Hide abstract] ABSTRACT: We have developed an assay based on rice embryogenic callus for the rapid functional characterization of metabolic genes. We validated the assay using a selection of well-characterized genes with known functions in the carotenoid biosynthesis pathway, allowing the rapid visual screening of callus phenotypes based on tissue color. We were then able to use the system to identify the functions of two uncharacterized genes: a chemically-synthesized β-carotene ketolase gene optimized for maize codon usage; and a wild-type Arabidopsis thaliana ortholog of the cauliflower Orange gene. In contrast to previous reports, we found that the wild-type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation could be induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm-specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the impact of metabolic engineering in complex pathways and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on the analysis and engineering of the carotenoid biosynthesis pathway. This article is protected by copyright. All rights reserved.
The Plant Journal 11/2013; 77(3). DOI:10.1111/tpj.12384 · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Over the last two decades, many carotenogenic genes have been cloned and used to generate metabolically-engineered plants producing higher levels of carotenoids. However, comparatively little is known about the regulation of endogenous carotenogenic genes in higher plants, and this restricts our ability to predict how engineered plants will perform in terms of carotenoid content and composition. During petal development in the Great Yellow Gentian (Gentiana lutea), carotenoid accumulation, the formation of chromoplasts and the upregulation of several carotenogenic genes are temporally coordinated. We investigated the regulatory mechanisms responsible for this coordinated expression by isolating five G. lutea carotenogenic gene (GlPDS, GlZDS, GlLYCB, GlBCH and GlLYCE) promoters by inverse PCR. Each promoter was sufficient for developmentally-regulated expression of the gusA reporter gene following transient expression in tomato (Solanum lycopersicum cv. Micro-Tom). Interestingly, the GlLYCB and GlBCH promoters drove high levels of gusA expression in chromoplast-containing mature green fruits, but low levels in chloroplast-containing immature green fruits, indicating a strict correlation between promoter activity, tomato fruit development and chromoplast differentiation. As well as core promoter elements such as TATA and CAAT boxes, all five promoters together with previously characterized GlZEP promoter contained three common cis-regulatory motifs involved in the response to methyl jasmonate (CGTCA) and ethylene (ATCTA), and required for endosperm expression (Skn-1_motif, GTCAT). These shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation. Our data provide insight into the regulatory basis of the coordinated upregulation of carotenogenic gene expression during flower development in G. lutea.
Physiologia Plantarum 11/2013; 150(4). DOI:10.1111/ppl.12129 · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metabolic engineering in plants can be used to increase the abundance of specific valuable metabolites, but single-point interventions generally do not improve the yields of target metabolites unless that product is immediately downstream of the intervention point and there is a plentiful supply of precursors. In many cases, an intervention is necessary at an early bottleneck, sometimes the first committed step in the pathway, but is often only successful in shifting the bottleneck downstream, sometimes also causing the accumulation of an undesirable metabolic intermediate. Occasionally it has been possible to induce multiple genes in a pathway by controlling the expression of a key regulator, such as a transcription factor, but this strategy is only possible if such master regulators exist and can be identified. A more robust approach is the simultaneous expression of multiple genes in the pathway, preferably representing every critical enzymatic step, therefore removing all bottlenecks and ensuring completely unrestricted metabolic flux. This approach requires the transfer of multiple enzyme-encoding genes to the recipient plant, which is achieved most efficiently if all genes are transferred at the same time. Here we review the state of the art in multigene transformation as applied to metabolic engineering in plants, highlighting some of the most significant recent advances in the field.
The International journal of developmental biology 10/2013; 57(6-7-8):565-576. DOI:10.1387/ijdb.130162pc · 1.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The biofortification of staple crops with vitamins is an attractive strategy to increase the nutritional quality of human food, particularly in areas where the population subsists on a cereal-based diet. Unlike other approaches, biofortification is sustainable and does not require anything more than a standard food-distribution infrastructure. The health-promoting effects of vitamins depend on overall intake and bioavailability, the latter influenced by food processing, absorption efficiency and the utilisation or retention of the vitamin in the body. The bioavailability of vitamins in nutritionally enriched foods should ideally be adjusted to achieve the dietary reference intake in a reasonable portion. Current vitamin biofortification programmes focus on the fat-soluble vitamins A and E, and the water-soluble vitamins C and B9 (folate), but the control of dosage and bioavailability has been largely overlooked. In the present review, we discuss the vitamin content of nutritionally enhanced foods developed by conventional breeding and genetic engineering, focusing on dosage and bioavailability. Although the biofortification of staple crops could potentially address micronutrient deficiency on a global scale, further research is required to develop effective strategies that match the bioavailability of vitamins to the requirements of the human diet.
Nutrition Research Reviews 10/2013; 26(02):1-11. DOI:10.1017/S0954422413000176 · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antibodies are an important class of proteins that can be used for the prevention, treatment and diagnosis of many diseases. Consequently, there is an intense and growing demand for recombinant antibodies, placing immense pressure on current production capacity which is based largely on microbial cultures and mammalian cells. Alternative systems for cost effective antibody production would be very welcome, and plants are now gaining widespread acceptance as green bioreactors with advantages in terms of cost, scalability and safety. Several plant-produced antibodies (plantibodies) are undergoing clinical trials and the first commercial approval could be only a few years away. The performance of the first generation of products has been very encouraging so far. In terms of product authenticity, differences in glycosylation between plantibodies and their mammalian counterparts have been defined, and the scientific evaluation of any possible consequences is underway. Ongoing studies are addressing the remaining biochemical constraints, and aim to further improve product yields, homogeneity and authenticity, particularly where the antibody is intended for injection into human patients. A remaining practical challenge is the implementation of large-scale production and processing under good manufacturing practice conditions that are yet to be endorsed by regulatory bodies. The current regulatory uncertainty and the associated costs represent an entry barrier for the pharmaceutical industry. However, the favourable properties of plants are likely to make the plant systems a useful alternative for small, medium and large scale production throughout the development of new antibody-based pharmaceuticals.