The Global Situation and Perspectives of Biotech Crops for the New Bioeconomy (Biofuels

The global situation and perspectives of biotech crops for the new bioeconomy (biofuels and bio-based chemicals)

Emilio Rodríguez-Cerezo

European Commission, Joint Research Centre (JRC), Edificio EXPO, C/Inca Garcilaso s/n

E-41092

Seville/Spain

+34 954488398

The conversion of renewable biological resources into value-added products, such as food, feed, bio-based chemicals and materials and energy is the definition of the Bioeconomy. In this presentation we shall focus on two important industrial sectors: the manufacturing of liquid biofuels and the production of bio-based chemicals. These two sectors use agricultural and/or forestry based feedstocks. In the first part we shall review the current crops used by the EU biofuel and bio-based chemical industries. These will include looking at the EU balance sheets for the crops producing the main raw materials: sugar, starch, oils, fats and cellulose. Next, we will look at the crops identified to analyse whether biotech crops are currently used for their production. We will describe the main biotech traits/crops used in the bioeconomy. Looking at the future, we will describe the projections for new feedstock needs for the bioeconomy (particularly to implement cellulosic biomass-derived processes) and will analyse how the pipeline of new biotech crops foreseen for 2020 will address these needs. Social Issues such as labelling and social acceptance of biotech crops in the bioeconomy will be discussed. The potential on new technologies, different from GM transformation, will also be discussed.

Plant genes and processes important for Agrobacterium-mediated genetic transformation

Stanton B. Gelvin

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 USA

Agrobacterium-mediated plant genetic transformation is a complex process involving both bacterial genes/proteins and host plant genes/proteins. Agrobacterium transfers single strand T-DNA (T-strands), piloted by VirD2 protein covalently linked to the 5’ end, and four other virulence effector proteins into the plant cell. Within the plant, T-DNA/VirD2 likely complexes with Agrobacterium VirE2 protein (a single-strand DNA binding protein), other bacterial Virulence effector proteins, and plant proteins. These complexes help traffic T-DNA through the cytoplasm to the nucleus, and target T-DNA to the plant genome for integration and transgene expression. My laboratory has used numerous approaches to identify plant genes/proteins involved in these various steps in transformation, including forward and reverse genetic screens to identify Arabidopsis mutants that are resistant or hypersusceptible to transformation, yeast two-hybrid and bimolecular fluorescence complementation assays to identify plant proteins that interact with Virulence effector proteins, and microarray and RNA-seq analyses to identify host genes whose expression is influenced by Agrobacterium or by the presence of specific Virulence effector proteins. Among these are plant cell wall proteins important for bacterial binding, importin- proteins important for nuclear trafficking of the T-strand, chromatin proteins important for T-DNA integration, and a myb transcription factor that acts as a global negative regulator of transformation susceptibility. I shall present these data with regard to answering the question “What makes plants susceptible or resistant to Agrobacterium-mediated transformation”?

Social implementation of vaccine rice for Japanese cedar pollinosis and development novel genome engineering technology

Yutaka TABEI

Institute of Agrobiological Sciences, NARO

Transgenic crops such as herbicide soybean and insect resistance corn had been cultivated from 1996 and new plant breeding technology (NPBT), especially genome editing, also is attracting worldwide attention. These techniques are effective breeding methods and it is expected that development of novel functional crops will accelerates. Here I introduce vaccine rice for Japanese cedar pollinosis and new genome editing procedure. The pollinosis is one of the most widespread diseases around the world. Japanese cedar (Cryptomeria japonica) pollinosis is a predominant allergic disease in Japan and prevalence is reported to be more than 25% of population. One is transgenic rice accumulating the 7Crp peptide that is composed of seven major human T-cell epitopes derived from major cedar pollen allergens Cry j 1 and Cry j 2. Clinical studies are conducting from 2016 at two medical institutes.

In order to perform genome editing in plants, it is necessary to introduce CRISPR / Cas9 gene once. We demonstrated that split-SaCas9 expressed transiently and separately from Tomato mosaic virus (ToMV) and Agrobacterium was able to direct targeted mutagenesis. Moreover two plant viruses harboring a split Cas9 system represents a promising tool for integration-free targeted mutagenesis in plant. These improvements due to fundamental research are expected to boost development of new varieties and social implementation of them.

From sex chromosomes to sex determination in Lepidoptera

Frantisek Marec

Institute of Entomology, Biology Centre CAS, Ceske Budejovice, Czech Republic;

Moths and butterflies (Lepidoptera) have sex chromosome systems with female heterogamety (WZ/ZZ or derived variants). However, the actual mechanism of sex determination is largely unknown. Only recently the primary sex-determining factor was discovered in the silkworm (Bombyx mori). In this model species, the W chromosome encodes the Fem piRNA, which promotes femaleness by downregulation of the expression of a Z-linked gene, Masculinizer, promoting male development in the W absence. However, little is known about the role of W chromosome in other Lepidoptera. The W chromosome is a novelty in Lepidoptera, as it is absent in the sister order Trichoptera (caddisflies) and in primitive moths such as Micropterigidae. Based on the W chromosome occurrence in lower Lepidoptera and deep conservation of the Z chromosome, we have revised the hypothesis of the origin of the W chromosome. Furthermore, our results in hybrids of wild silkmoths (Samia cynthia) questioned the conserved role of the W chromosome in determining female sex. To facilitate sequence analysis of W chromosomes, we have developed a straightforward strategy based on laser microdissection of W chromatin, which enabled us to produce a high quality PacBio W-chromosome assembly in the diamondback moth (Plutella xylostella). We have also identified several genes of the silkworm sex-determining pathway in the flour moth (Ephestia kuehniella). The function of these genes is currently being tested.

Revolutionize the silk industry through the use of GM technology

Keiko Kadono-Okuda

Institute of Agrobiological Sciences, NARO

In 2000, a team led by Dr. Tamura succeeded in genetically engineering the silkworm, Bombyx mori. The study of transgenic silkworms has since developed the means of producing unique silk textiles and medical products. It is important to pursue such research in order to revolutionize the silk industry; to create new industries and markets, and revitalize local businesses. In our institute, we have been developing a variety of transgenic silkworm lines designed to produce fluorescent-colored silk, spider silk, ultra-thin silk with high dye affinity, antibodies, diagnostic reagents and proteins, etc. To expand the possibilities of transgenic silkworms and to enter a new era of sericulture, we aim to construct efficient systems for producing recombinant proteins, to develop new silk materials using transgenic silkworms, and to commercialize the processed silk as new materials.

GAL4/UAS system was employed for the recombinant protein production. The system enabled the silkworm to produce a wide variety of useful proteins; some of these recombinant proteins or the products containing these recombinant proteins are commercially available. One of the successful expressions is the anti-CD20 monoclonal antibody, Rituximab, which is used in medicine for the treatment hematological cancers and autoimmune diseases. The antibody produced in the transgenic silkworm has stronger antibody-dependent cell-mediated cytotoxicity (ADCC) compared with the antibody produced in Chinese hamster ovary (CHO) cells. To improve the genome engineering technology of the silkworm, it is critical to develop a new method of genome editing utilising TALEN, CRISPR/Cas9 and homologous recombination.

In order to promote the production of recombinant silk such as fluorescent or ultra-thin silk, farmers needed to be involved for rearing transgenic silkworm, for which we needed approval for Type 1 Use. For this purpose, we had been accumulating and analyzing scientific data for the evaluation of biological diversity under Cartagena Act. This year, Type 1 Use of the GFP-silkworm was approved as a first living modified animal in Japan on September 22, and farm-rearing of the GFP-silkworms was started on October 5. We are now on the way to the new silk industry through the use of GM technology.

Strategies to produce multi-transgenic pigs for xenotransplantation

Heiner Niemann, 1Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, Neustadt a. Rbge., Germany, 2 REBIRTH, Cluster of Excellence, Hannover Medical School.

Allotransplantation is the only effective therapy for end-stage human organ failure. However, many patients in need do not receive a suitable organ owing to the ever growing shortage of human organ donors. More than 60,000 registered patients are currently waiting for a life-saving organ in the European Union. Thus, functional porcine xenografts and cells to treat patients with terminal organ failure would be of tremendous value for human medicine. The domestic pig shares many genetic, anatomical and physiological similarities to humans and the ability to genetically modify pigs significantly enhances their potential as organ donor. Moreover, the discovery of new molecular tools to modify complex mammalian genomes such as ZFNs, TALENs and CRISPR/Cas, and the improved genomic maps of human and pigs open new avenues towards efficient, precise and fast modification of the porcine genome. However, prior to clinical application of porcine xenografts, three major hurdles have to be overcome: (i) various immunological rejection responses, incl. HAR (hyperacute rejection response), AVR (acute vascular rejection), DXR (delayed cellular rejection), (ii) physiological incompatibilities between the porcine organ and the human recipient, incl. a severe dysfunction of the coagulation cascade, and (iii) the risk of transmitting zoonotic pathogens from pig to humans.

The generation of pigs with a genetic knockout of the ɑ1.3-galactosyltransferase gene (GGTA1) was a milestone down the road towards clinical application of porcine xenografts. The HAR can now be reliably prevented and significantly extended survival times after pig-to-baboon xenotransplantation up to a maximum of 83 days for kidneys and more than two years for heterotopically transplanted hearts have been reported. After orthotopic (i.e. life supportive) heart transplants the average survival of the recipient is 30-50 days, with a maximum of 90 days. Subsequently porcine xenografts are rejected due to inflammatory symptoms and severe perturbation of coagulation. Thus, the AVR remains the bottleneck to clinical xenotransplantation. Non-anti-Gal antibody binding activates the endothelium and results in cellular damage and thrombotic microangiopathy. The current view is that long-term survival of xenografts after transplantation into primates requires multiple modifications of the porcine genome and a specifically tailored immunosuppressive regimen compliant with current clinical standards. This requires the production and characterization of multi-transgenic pigs to control HAR, AVR and DXR. Several candidate genes, incl. hTM, hHO-1, hA20, CTLA4Ig, have been explored in their ability to improve long-term survival of porcine xenografts after transplantation into non-human primates. The presentation will provide an update on the current status in the production of multi-transgenic pigs for xenotransplantation, with emphasis on recent results from our laboratory, which could bring porcine xenografts closer to clinical application.

Selected own references

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